U.S. patent number 11,174,511 [Application Number 16/633,763] was granted by the patent office on 2021-11-16 for methods and compositions for selecting and amplifying dna targets in a single reaction mixture.
This patent grant is currently assigned to Dana-Farber Cancer Institute, Inc.. The grantee listed for this patent is Dana-Farber Cancer Institute, Inc.. Invention is credited to Gerassimos Makrigiorgos.
United States Patent |
11,174,511 |
Makrigiorgos |
November 16, 2021 |
Methods and compositions for selecting and amplifying DNA targets
in a single reaction mixture
Abstract
This disclosure relates to compositions and methods for
single-step, multi-stage amplification reactions that combine many
stages of sample preparation process in a single tube reaction. The
disclosed technology provides a mean of performing multiplexed
nested PCR in a single vessel, without any need of purification
steps, and is based on the use of three sets of primers: a pair of
outer primers, a pair of inner primers that are nested within the
pair of outer primers, and tail primers that are complementary to
tails on the inner primers. By adjusting the temperature
conditions, annealing temperatures of the primers, number of
amplification cycles, and the concentrations of the outer, inner,
and tail primers, it is possible to carry out multiplexed nested
PCR in a single vessel.
Inventors: |
Makrigiorgos; Gerassimos
(Chestnut Hill, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Dana-Farber Cancer Institute, Inc. |
Boston |
MA |
US |
|
|
Assignee: |
Dana-Farber Cancer Institute,
Inc. (Boston, MA)
|
Family
ID: |
65039768 |
Appl.
No.: |
16/633,763 |
Filed: |
July 24, 2018 |
PCT
Filed: |
July 24, 2018 |
PCT No.: |
PCT/US2018/043506 |
371(c)(1),(2),(4) Date: |
January 24, 2020 |
PCT
Pub. No.: |
WO2019/023243 |
PCT
Pub. Date: |
January 31, 2019 |
Prior Publication Data
|
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|
|
Document
Identifier |
Publication Date |
|
US 20200370108 A1 |
Nov 26, 2020 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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62536187 |
Jul 24, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6844 (20130101); C12Q 1/6848 (20130101); C12Q
1/6848 (20130101); C12Q 2525/155 (20130101); C12Q
2527/101 (20130101); C12Q 2537/143 (20130101); C12Q
2547/101 (20130101); C12Q 2549/113 (20130101); C12Q
1/6844 (20130101); C12Q 2525/155 (20130101); C12Q
2527/101 (20130101); C12Q 2537/143 (20130101); C12Q
2547/101 (20130101); C12Q 2549/113 (20130101) |
Current International
Class: |
C12Q
1/68 (20180101); C12Q 1/6848 (20180101) |
Field of
Search: |
;435/6.12 |
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|
Primary Examiner: Priest; Aaron A
Attorney, Agent or Firm: Wolf, Greenfield & Sacks,
P.C.
Parent Case Text
RELATED APPLICATIONS
This Application is a national stage filing under 35 U.S.C. 371 of
International Patent Application Serial No. PCT/US2018/043506,
filed Jul. 24, 2018, which claims the benefit under 35 USC 119(e)
of U.S. provisional patent application Ser. No. 62/536,187, filed
Jul. 24, 2017, the entire contents of each of which are
incorporated by reference herein.
Claims
The invention claimed is:
1. A method of selecting and amplifying DNA targets in a single
reaction vessel, the method comprising the following steps: (a)
providing in the single reaction vessel: a sample of
double-stranded DNA, a set of outer multiplexed primers comprising
an outer forward primer and an outer reverse primer, wherein each
of the outer forward and reverse primers complement target nucleic
acids on the DNA, and wherein the concentration of the outer
multiplexed primers is 0.01-0.2 .mu.M, a set of inner multiplexed
primers comprising an inner forward primer and an inner reverse
primer, wherein each of the inner forward and reverse primers
comprises a target-specific anchor on its 3' end, and the inner
forward primer comprises a common forward tail on its 5' end and
the inner reverse primer comprises a common reverse tail on its 5'
end, wherein the common forward tail is different from the common
reverse tail, and wherein the concentration of the inner
multiplexed primers is 0.001-0.04 .mu.M, a set of tail primers
comprising of a first tail primer and a second tail primer, wherein
the first tail primer is complementary to the common forward tail
and the second tail primer is complementary to the common reverse
tail, and wherein the concentration of the tail primers is 0.1-1
.mu.M, wherein the outer multiplexed primers have an annealing
temperature of 60-65.degree. C., and the inner multiplexed primers
have an annealing temperature of 50-55.degree. C., or wherein the
outer multiplexed primers have an annealing temperature of
58-62.degree. C., and the inner multiplexed primers have an
annealing temperature of 66-70.degree. C.; (b) subjecting the
provided contents in the single reaction vessel to an amplification
condition which is carried out for 8-12 cycles and favors the
annealing of the set of outer multiplexed primers to the DNA; (c)
subjecting the provided contents in the single reaction vessel to
an amplification condition which is carried out for 2-6 cycles and
favors annealing of the set of inner multiplexed primers to
amplified products of step (b); and (d) subjecting the provided
contents in the single reaction vessel to an amplification
condition which is carried out for 10-30 cycles and under which the
set of tail primers anneal to the amplified products of step
(c).
2. The method of claim 1, further providing in the single reaction
vessel a DNA polymerase, dNTPs and an amplification buffer.
3. The method of claim 1, wherein the annealing temperature of the
inner multiplexed primers is 5-15.degree. C. lower or 5-10.degree.
C. higher than the annealing temperature of the outer multiplexed
primers.
4. The method of claim 1, wherein the annealing temperature of the
tail primers is 3-20.degree. C. lower or higher than the annealing
temperature of the inner multiplexed primers.
5. The method of claim 4, wherein the annealing temperature of the
tail primers is 60-70.degree. C.
6. The method of claim 1, wherein the inner multiplexed primers are
hot start primers, activated by subjecting the provided contents in
the single reaction vessel to an activation temperature after the
completion of step (b) or step (c).
7. The method of claim 6, wherein the activation temperature is
90-95.degree. C., and wherein the provided contents in the single
reaction vessel is subjected to the activation temperature for 5
seconds to 5 minutes.
8. The method of claim 1, wherein the ratio of the concentration of
outer multiplexed primers to the concentration of inner multiplexed
primers is 0.25-2000, the ratio of the concentration of the tail
primers to the concentration of inner multiplexed primers is 5-200,
or the ratio of the concentration of the tail primer to the
concentration of the outer multiplexed primers is 1-20.
9. The method of claim 1, wherein the number of amplification
cycles in step (d) exceeds the number of amplification cycles in
step (b).
10. The method of claim 1, wherein the tail primers further
comprise 20-30 bp of a 3'end portion of a sequencing adapter.
11. The method of claim 1, wherein the inner forward and inner
reverse primers each further comprise, between the target-specific
anchor and the common forward or reverse tails, a central portion
that is a unique barcode.
12. The method of claim 11, wherein the inner multiplexed primers
are provided such that the ratio of DNA to unique barcodes is
10.sup.7-10.sup.9 unique barcodes to 100 ng DNA.
13. The method of claim 1, further comprising enriching mutant
alleles of the target nucleic acids relative to wild-type alleles
of the target nucleic acids after the completion of step (d).
14. The method of claim 13, wherein the enriching the mutant
alleles of the target regions relative to wild-type alleles of the
target nucleic acids comprises subjecting the provided contents in
the single reaction vessel after completion of step (d) to
Nuclease-assisted Minor-allele Enrichment using Probe Overlap
(NaME-PrO), Coamplification at Lower Denaturation temperature-PCR
(COLD-PCR), Improved and Complete Enrichment COLD-PCR
(ice-COLD-PCR), Temperature-Tolerant-ice-COLD-PCR
(TT-ice-COLD-PCR), toehold PCR, or Differential Strand Separation
at Critical Temperature (DiSSECT).
15. The method of claim 1, wherein step (d) comprises one or more
of the following: Coamplification at Lower Denaturation
temperature-PCR (COLD-PCR), Improved and Complete Enrichment
COLD-PCR (ice-COLD-PCR), Temperature-Tolerant-ice-COLD-PCR
(TT-ice-COLD-PCR), and toehold PCR.
16. A reaction mixture comprising a set of outer multiplexed
primers that complement target nucleic acids on DNA, a set of outer
multiplexed primers at a concentration of 0.01-0.2 .mu.M comprising
an outer forward primer and an outer reverse primer, wherein each
of the outer forward and reverse primers complement target nucleic
acids on the DNA, a set of inner multiplexed primers at a
concentration of 0.001-0.04 .mu.M comprising an inner forward
primer and an inner reverse primer, wherein each of the inner
forward and reverse primers comprises a target-specific anchor on
its 3' end, and the inner forward primer comprises a common forward
tail on its 5' end and the inner reverse primer comprises a common
reverse tail on its 5' end, wherein the common forward tail is
different from the common reverse tail, a set of tail primers at a
concentration of 0.1-1 .mu.M comprising of a first tail primer and
a second tail primer, wherein the first tail primer is
complementary to the common forward tail and the second tail primer
is complementary to the common reverse tail, wherein the outer
multiplexed primers have an annealing temperature of 60-65.degree.
C., and the inner multiplexed primers have an annealing temperature
of 50-55.degree. C., or wherein the outer multiplexed primers have
an annealing temperature of 58-62.degree. C., and the inner
multiplexed primers have an annealing temperature of 66-70.degree.
C.
17. A reaction mixture comprising a set of outer multiplexed
primers at a concentration of 0.01-0.2 .mu.M comprising an outer
forward primer and an outer reverse primer, wherein (i) the outer
forward primer complements a common tag and the outer reverse
primer complements target nucleic acids on DNA, or (ii) the outer
reverse primer complements a common tag and the outer forward
primer complements target nucleic acids on DNA, a set of inner
multiplexed primers at a concentration of 0.001-0.04 .mu.M
comprising an inner forward primer and an inner reverse primer,
wherein (H) the inner forward primer is complementary to the common
tag, which comprises a common forward tail, and wherein the inner
reverse primer comprises a target-specific anchor on its 3' end and
a common reverse tail on its 5' end, or (ii) the inner reverse
primer is complementary to the common tag, which comprises a common
reverse tail, and wherein the inner forward primer comprises a
target-specific anchor on its 3' end and a common forward tail on
its 5' end, wherein the common forward tail is different from the
common reverse tail, and a set of tail primers at a concentration
of 0.1-1 .mu.M comprising of a first tail primer and a second tail
primer, wherein the first tail primer is complementary to the
common forward tail and the second tail primer is complementary to
the common reverse tail, wherein the outer multiplexed primers have
an annealing temperature of 60-65.degree. C., and the inner
multiplexed primers have an annealing temperature of 50-55.degree.
C., or wherein the outer multiplexed primers have an annealing
temperature of 58-62.degree. C., and the inner multiplexed primers
have an annealing temperature of 66-70.degree. C.
Description
BACKGROUND OF THE INVENTION
Next generation sequencing (NGS) is currently widely employed in
the field of personalized medicine to derive the molecular profile
of human specimens such as those obtained from cancer biopsies,
blood, urine or other excretions. Molecular profiling of patient
specimens can have broad diagnostic, prognostic or predictive
information for the course of diseases like cancer. Preparation of
patient specimens for NGS and targeted re-sequencing usually
involves several steps that can last several days, thus increasing
the time to obtaining results which can be important to the
patients and their treatments. Longer sample preparation times also
unavoidably result in higher costs.
SUMMARY OF THE INVENTION
This disclosure relates to compositions and methods for
single-step, multi-stage amplification reactions that combine many
stages of sample preparation process in a single tube reaction. The
disclosed technology provides a mean of performing multiplexed
nested PCR in a single vessel, without any need of purification
steps. In some embodiments, the rapid and streamlined sample
preparation methods result in highly specific coverage of the
targeted DNA sites, thus improving the efficiency of NGS and
reducing costs. Such multi-stage PCR reactions may optionally
include enrichment steps like COLD-PCR or NaME-PrO. The disclosed
compositions and methods are also particularly suited for sensitive
and effective sequencing of low-level or less abundant mutations
via the incorporation of molecular barcodes.
The present disclosure is based on the discovery that serial
amplification reactions can be carried out in a single vessel when
one understands and takes advantage of the subtle relationship
between temperature conditions, concentration of oligonucleotides,
and annealing and melting temperature (typically determined by
length) of oligonucleotides. The disclosed technology is based on
the use of three sets of primers: a pair of outer primers, a pair
of inner primers that are nested within the pair of outer primers,
and tail primers that are complementary to tails on the inner
primers. By adjusting the temperature conditions, annealing
temperatures of the primers, number of amplification cycles, and
the concentrations of the outer, inner, and tail primers, it is
possible to carry out multiplexed nested PCR in a single vessel.
See for example, FIG. 1. In summary, one can selectively drive
amplification through any one of the three sets of primers, as
desired.
One way to implement the ability to selectively amplify is based
primarily on varying the concentration and annealing temperatures
of the primers. In one such embodiment, the concentration of the
outer primers is lower than, or in the range of primer
concentrations that are typically used in PCR (e.g., 0.01-0.2
.mu.M). The concentration of the inner primers is relatively much
lower than the concentration of the outer primers, such that
carrying out amplification cycles at the annealing temperature of
the outer primers favors outer primer binding due to the greater
concentration of the outer primers. The inner primer concentration
is lower than the outer primer concentration, such that if the
outer primers were absent, or if the first step of amplification of
any one of the methods described herein were not performed, then
amplification using the inner primers would yield only a relatively
insignificant amount of product from the DNA sample (e.g., about
100 times less, 1000 times less, 10,000 times less, or 100,000
times less). When amplifying at temperatures favorable to inner
primer binding but above a temperature favorable to outer primer
binding, one achieves amplification driven by inner primer binding.
The inner primers, which are nested within the outer primers,
provide another level of specificity to any one of the disclosed
methods herein. Finally, the bulk of the amplification can occur by
using tail primers with annealing temperatures above those for the
outer and inner primers, driving amplification solely through the
tail primers.
Provided in the single reaction vessel are (i) a sample of
double-stranded DNA (e.g., genomic DNA, or cDNA); (ii) a set of
outer multiplexed primers; (iii) a set of inner multiplexed
primers; and (iv) a set of tail primers. The kinetic conditions of
the reaction can be altered to favor the annealing of the set of
outer multiplexed primers over the set of inner multiplexed
primers. For example, if both sets of primers are the same length
and present at the same concentration, and have the same melting
temperature (Tm) and annealing temperature (Ta), the primers will
anneal similarly. However, if the set of outer multiplexed primers
are present in an excess (e.g., 10.times. excess) compared to the
inner multiplexed primers, they will anneal more favorably than the
set of inner multiplexed primers. If the set of outer multiplexed
primers are longer than the set of inner multiplexed primers and
their annealing temperature is above the temperature that allows
the set of inner multiplexed primers to anneal, this will favor
annealing of the set of outer multiplexed primers at temperatures
above the annealing temperature of the inner multiplexed primers.
Once the PCR product generated by the outer multiplexed primers
starts building up, the increased concentration of the amplified
region will now offer ample template for the inner primers to also
bind substantially and generate PCR product which is nested to the
product produced by the outer primers, and thereby being highly
specific to the intended DNA targets. Finally, the set of tail
primers will anneal after the set of outer multiplexed primers and
after the set of inner multiplexed primers because the tail
sequence is not present in the template DNA until after the
amplification reaction with the inner multiplexed primers. The tail
primers can be selected, for example, to be short with a relatively
low annealing temperature but at a relatively high concentration,
such that conditions can be applied to favor binding of the tail
primer.
In another embodiment, the outer multiplex primers are at a
concentration that is lower than the inner multiplex primers and
the outer multiplex primers have an annealing temperature that is
higher than that of the inner multiplex primers. If temperatures
are applied at the annealing temperature of the outer multiplex
primers (above the annealing temperature of the inner multiplex
primers), then the outer multiplex primers will anneal and extend.
Following repeated such extensions, then the temperature can be
brought to the annealing temperature of the inner multiplex primers
(which is lower than the annealing temperature of the outer
multiplex primers) and these conditions will favor the annealing of
the inner multiplex primers because the inner multiplex primers are
at a higher concentration than the outer multiplex primers.
Following repeated such extensions, then the tail primers may be
annealed and extended. If the tail primers are at a higher
concentration and have a lower annealing temperature than the inner
and outer multiplex primers, then the temperature can be brought to
the tail primer annealing temperature, and these conditions will
favor the annealing of the tail primers because the tail primers
are at a higher concentration than the inner and outer multiplex
primers.
It therefore can be understood that the outer multiplexed primers,
the inner multiplexed primers and the tail primers can be in the
same vessel, and the various reactions (first outer primer
extension, then inner primer extension, and then tail primer
extension) can be carried out in the desired order in that single
vessel, based on selecting appropriate primers, concentrations
(e.g., primer concentrations) and temperature conditions. This
provides extraordinary advantages over the procedures of the prior
art.
In one aspect, provided are methods for selecting and amplifying
DNA targets in a single reaction vessel. The methods comprise the
following steps: (a) providing in the single reaction vessel: a
sample of double-stranded DNA (e.g., genomic DNA, or cDNA), a set
of outer multiplexed primers comprising an outer forward primer and
an outer reverse primer, wherein each of the outer forward and
reverse primers complement target nucleic acids on the DNA, a set
of inner multiplexed primers comprising an inner forward primer and
an inner reverse primer, wherein each of the inner forward and
reverse primers comprises a target-specific anchor on its 3' end,
and the inner forward primer comprises a common forward tail on its
5' end and the inner reverse primer comprises a common reverse tail
on its 5' end, wherein the common forward tail is different from
the common reverse tail, a set of tail primers comprising of a
first tail primer and a second tail primer, wherein the first tail
primer is complementary to the common forward tail and the second
tail primer is complementary to the common reverse tail; (b)
subjecting the provided contents in the single reaction vessel to
an amplification condition which favors the annealing of the set of
outer multiplexed primers to the DNA; (c) subjecting the provided
contents in the single reaction vessel to an amplification
condition which favors annealing of the set of inner multiplexed
primers to amplified products of step (b); and (d) subjecting the
provided contents in the single reaction vessel to an amplification
condition under which the set of tail primers anneal to the
amplified products of step (c).
In another aspect, provided are methods of selecting and amplifying
DNA targets in a single reaction vessel. The methods comprise the
following steps: (a) providing in the single reaction vessel: a
sample of fragmented double-stranded DNA (e.g., genomic DNA, or
cDNA) comprising a unique identifier and a common tag at the 5' end
and at the 3' end, a set of outer multiplexed primers comprising an
outer forward primer and an outer reverse primer, (i) wherein the
outer forward primer complements the common tag and the outer
reverse primer complements target nucleic acids on the DNA, or (ii)
the outer reverse primer complements the common tag and the outer
forward primer complements target nucleic acids on the DNA, a set
of inner multiplexed primers comprising an inner forward primer and
an inner reverse primer, wherein (ii) the inner forward primer is
complementary to the common tag, which comprises a common forward
tail, and wherein the inner reverse primer comprises a
target-specific anchor on its 3' end and a common reverse tail on
its 5' end, or (ii) the inner reverse primer is complementary to
the common tag, which comprises a common reverse tail, and wherein
the inner forward primer comprises a target-specific anchor on its
3' end and a common forward tail on its 5' end, wherein the common
forward tail is different from the common reverse tail, a set of
tail primers comprising of a first tail primer and a second tail
primer, wherein the first tail primer is complementary to the
common forward tail and the second tail primer is complementary to
the common reverse tail; (b) subjecting the provided contents in
the single reaction vessel to an amplification condition which
favors the annealing of the set of outer multiplexed primers to the
DNA; (c) subjecting the provided contents in the single reaction
vessel to an amplification condition which favors annealing of the
set of inner multiplexed primers to amplified products of step (b);
and (d) subjecting the provided contents in the single reaction
vessel to an amplification condition under which the set of tail
primers anneal to the amplified products of step (c).
As used herein, "selection of DNA targets or target sequences"
means picking out target DNA sequences to amplify. A DNA target may
be selected on the basis of a known region of mutation, or to
search for an unknown mutation in a DNA sample. For example, a
particular DNA sequence may be targeted for determine the presence
of a particular mutation that may cause, or aid in the diagnosis of
a particular disease. In some embodiments, a DNA target is selected
as a control. By virtue of selecting a sequence of particular
consecutive base pairs in a DNA and performing any one of the
methods disclosed herein, one is selectively amplifying that
sequence.
In some embodiments, the methods further comprise providing in the
single reaction vessel a DNA polymerase, dNTPs and an amplification
buffer.
In some embodiments, annealing temperatures of the inner
multiplexed primers is 3-20.degree. C. (e.g., 3-5, 3-10, 5-10,
5-15, 5-20, or 10-20.degree. C.) different from annealing
temperature of the outer multiplexed primers. In some embodiments,
annealing temperatures of the inner multiplexed primers is
3-20.degree. C. (e.g., 3-5, 3-10, 5-10, 5-15, 5-20, or
10-20.degree. C.) lower than the annealing temperature of the outer
multiplexed primers. In some embodiments, the annealing temperature
of the outer multiplexed primers is 60-65.degree. C., and the
annealing temperature of the inner multiplexed primers is
50-55.degree. C. In some embodiments, the annealing temperatures of
the inner multiplexed primers is 3-20.degree. C. (e.g., 3-5, 3-10,
5-10, 5-15, 5-20, or 10-20.degree. C.) higher than the annealing
temperature of the outer multiplexed primers. In some embodiments,
the annealing temperature of the outer multiplexed primers is
58-62.degree. C., and the annealing temperature of the inner
multiplexed primers is 66-70.degree. C. In some embodiments, the
annealing temperature of the tail primers is 3-20.degree. C. (e.g.,
3-5, 3-10, 5-10, 5-15, 5-20, or 10-20.degree. C.) different from
the annealing temperature of the inner multiplexed primers. In some
embodiments, the annealing temperature of the tail primers is
60-70.degree. C.
In some embodiments, the inner multiplexed primers are hot start
primers, activated by subjecting the provided contents in the
single reaction vessel to an activation temperature after the
completion of step (b). In some embodiments, the tail primers are
hot start primers, activated by subjecting the provided contents in
the single reaction vessel to an activation temperature after the
completion of step (c). In some embodiments, the activation
temperature is 90-95.degree. C. In certain embodiments, the
provided contents in the single reaction vessel is subjected to an
activation temperature for 5 seconds to 5 minutes.
In some embodiments, the concentration of outer multiplexed primers
is 0.01-0.2 .mu.M. In certain embodiments, the concentration of
inner multiplexed primers is 0.001-0.04 .mu.M. In some embodiments,
the concentration of tail primers is 0.1-1 .mu.M. In some
embodiments, the ratio of concentration of outer multiplexed
primers to the concentration of inner multiplexed primers is
0.25-2000. In certain embodiments, the ratio of the concentration
of the tail primers to the concentration of inner multiplexed
primers is 5:200. In some embodiments, the ratio of the
concentration of the tail primer to the concentration of the outer
multiplexed primers is 1-20.
In some embodiments, the amplification of step (b) is carried out
for 8-12 cycles. In certain embodiments, the amplification of step
(c) is carried out for 2-6 cycles. In some embodiments, the
amplification of step (d) is carried out for 10-30 cycles. In
certain embodiments, the number of amplification cycles in step (b)
exceeds the number of amplification cycles in step (c). In some
embodiments, the number of amplification cycles in step (d) exceeds
the number of amplification cycles in step (c). In certain
embodiments, the number of amplification cycles in step (d) exceeds
the number of amplification cycles in step (b).
In some embodiments, the tail primers further comprise 20-30 bp of
a 3'end portion of a sequencing adapter. In certain embodiments,
the inner forward and inner reverse primers each further comprise,
between the target-specific anchor and the common forward or
reverse tails, a central portion that is a unique barcode. In some
embodiments, the unique barcode is 8-14 bp in length. In some
embodiments, the inner multiplexed primers are provided such that
the ratio of DNA (e.g., genomic DNA, or cDNA) to unique barcodes is
10.sup.7-10.sup.9 unique barcodes to 100 ng DNA.
In some embodiments, the methods further comprise enriching mutant
alleles of the target nucleic acids relative to wild-type alleles
of the target nucleic acids after the completion of step (d). In
some embodiments, the enriching the mutant alleles of the target
regions relative to wild-type alleles of the target nucleic acids
comprises subjecting the provided contents in the single reaction
vessel after the completion of step (d) to one or more of the
following: Nuclease-assisted Minor-allele Enrichment using Probe
Overlap (NaME-PrO), Coamplification at Lower Denaturation
temperature-PCR (COLD-PCR), Improved and Complete Enrichment
COLD-PCR (ice-COLD-PCR), Temperature-Tolerant-ice-COLD-PCR
(TT-ice-COLD-PCR), toehold PCR and Differential Strand Separation
at Critical Temperature (DiSSECT). In some embodiments, step (d)
comprises one or more of the following: Coamplification at Lower
Denaturation temperature-PCR (COLD-PCR), Improved and Complete
Enrichment COLD-PCR (ice-COLD-PCR),
Temperature-Tolerant-ice-COLD-PCR (TT-ice-COLD-PCR), and toehold
PCR.
In some embodiments, step (d) comprises subjecting the provided
contents in the single reaction vessel to one or more of the
following: Coamplification at Lower Denaturation temperature-PCR
(COLD-PCR), Improved and Complete Enrichment COLD-PCR
(ice-COLD-PCR), and Temperature-Tolerant-ice-COLD-PCR
(TT-ice-COLD-PCR). In some embodiments, COLD-PCR, ice-COLD-PCR,
TT-ice-COLD-PCR, or toehold PCR comprise the last amplification
cycles of step (d). In some embodiments, at least the first four
(e.g., 4, 5, 6, 7, 8, 9, 10, or more) amplification cycles of step
(d) do not comprise COLD-PCR, ice-COLD-PCR, TT-ice-COLD-PCR, or
toehold PCR. For example, step (d) may comprise four amplification
cycles of normal PCR followed by 10 cycles of COLD-PCR using tail
primers.
In some embodiments, the enrichment of mutant alleles of the target
nucleic acids relative to wild-type alleles of the target nucleic
acids by Coamplification at Lower Denaturation temperature-PCR
(COLD-PCR), Improved and Complete Enrichment COLD-PCR
(ice-COLD-PCR), or Temperature-Tolerant-ice-COLD-PCR
(TT-ice-COLD-PCR) is performed in the same tube in which steps (a),
(b), (c) and (d) were performed. This is only possible for
COLD-PCR, ice-COLD-PCR, TT-ice-COLD-PCR, or toehold PCR. For
NAME-PRO and DISSECT it has to be separate steps than step (d). In
further embodiments, the reagents for the enrichment of mutant
alleles of the target nucleic acids relative to wild-type alleles
of the target nucleic acids are provided in step (a).
In some embodiments, DNA is obtained from a biological sample. In
some embodiments, the biological sample is selected from the group
consisting of: tissue, blood, plasma, serum, urine, saliva and
cerebrospinal fluid. In some embodiments, the biological sample is
fixed or frozen. In certain embodiments, the biological sample is
formalin-fixed paraffin-embedded (FFPE).
In another aspect, provided are reaction mixtures. The reaction
mixtures comprise: a set of outer multiplexed primers that
complement target nucleic acids on DNA (e.g., genomic DNA, or
cDNA), a set of outer multiplexed primers comprising an outer
forward primer and an outer reverse primer, wherein each of the
outer forward and reverse primers complement target nucleic acids
on the DNA, a set of inner multiplexed primers comprising an inner
forward primer and an inner reverse primer, wherein each of the
inner forward and reverse primers comprises a target-specific
anchor on its 3' end, and the inner forward primer comprises a
common forward tail on its 5' end and the inner reverse primer
comprises a common reverse tail on its 5' end, wherein the common
forward tail is different from the common reverse tail, a set of
tail primers comprising of a first tail primer and a second tail
primer, wherein the first tail primer is complementary to the
common forward tail and the second tail primer is complementary to
the common reverse tail.
In another aspect, provided are reaction mixtures. The reaction
mixtures comprise: a set of outer multiplexed primers comprising an
outer forward primer and an outer reverse primer, (i) wherein the
outer forward primer complements a common tag and the outer reverse
primer complements target nucleic acids on DNA (e.g., genomic DNA,
or cDNA), or (ii) the outer reverse primer complements a common tag
and the outer forward primer complements target nucleic acids on
DNA, a set of inner multiplexed primers comprising an inner forward
primer and an inner reverse primer, wherein (ii) the inner forward
primer is complementary to the common tag, which comprises a common
forward tail, and wherein the inner reverse primer comprises a
target-specific anchor on its 3' end and a common reverse tail on
its 5' end, or (ii) the inner reverse primer is complementary to
the common tag, which comprises a common reverse tail, and wherein
the inner forward primer comprises a target-specific anchor on its
3' end and a common forward tail on its 5' end, wherein the common
forward tail is different from the common reverse tail, and a set
of tail primers comprising of a first tail primer and a second tail
primer, wherein the first tail primer is complementary to the
common forward tail and the second tail primer is complementary to
the common reverse tail.
In some embodiments, the outer forward primer, the inner forward
primer, and the first tail primer are common for all target nucleic
acids. In some embodiments, the outer reverse primer, the inner
reverse primer, and the second tail primer are common for all
target nucleic acids. In further embodiments, the primers that are
common for all targets are hot start primers.
Sequences of any double-stranded DNA can be amplified in the
methods disclosed herein. In some embodiments, double-stranded DNA
is genomic DNA. In some embodiments, double-stranded DNA is cDNA.
Genomic DNA can be sourced or obtained from a biological sample
(e.g., circulating DNA, a sample of tissue (e.g., a fixed or frozen
sample of tissue), urine, blood, plasma, serum, saliva, or
cerebrospinal fluid). In some embodiments, the reaction mixtures
further comprise a sample of DNA (e.g., genomic DNA, or cDNA). In
some embodiments, the DNA is sourced from a biological sample
sourced from tissue, blood, plasma, serum, urine, saliva or
cerebrospinal fluid. In some embodiments, the biological sample is
fixed or frozen.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and
are included to further demonstrate certain aspects of the present
disclosure, which can be better understood by reference to one or
more of these drawings in combination with the detailed description
of specific embodiments presented herein. It is to be understood
that the data illustrated in the drawings in no way limit the scope
of the disclosure.
FIG. 1 describes a single tube, all-in-one reaction to select DNA
targets of interest and add the sequencing adaptors in a single
multi-stage PCR. Majority of the final PCR product is generated by
the Tail oligonucleotides, which are at higher concentration than
other oligonucleotides in the reaction.
FIG. 2 shows a comparison of the single-tube multi-step PCR
approach (in black) with the established, multi-step PCR sample
preparation method (in white) for 54 DNA targets amplified from
genomic cfDNA simultaneously. The number of Miseq reads (total
counts) follows the same general trends for both approaches, thus
validating the single tube approach.
FIG. 3 describes single step `all in one` reaction incorporating
target selection as well as mutation enrichment via
COLD-PCR/ICE-COLD-PCR.
FIG. 4 depicts PCR cycling conditions applied for single-tube,
all-in-one reaction for two targets BRAF and KRAS amplified
directly from genomic DNA in one reaction, including also mutation
enrichment for both targets.
FIG. 5A shows a melting curve analysis of the single-tube
multiplexed all-in-one ICE-COLD-PCR reaction for BRAF and KRAS.
FIGS. 5B and 5C show digital PCR performed for KRAS that
demonstrates mutation enrichment during the single tube reaction. A
serial mutation dilution experiment was performed, where KRAS and
BRAF mutations were diluted at known, decreasing amounts. The
resulting mutation enrichment is inferred by the fractional
abundance detected via digital PCR (left axis). For example, the
1:10 sample has an original mutation abundance of about 0.35%,
which becomes 12.6% after the single-tube reaction. This indicates
an approximate 36-fold mutation enrichment.
FIGS. 6A-6B are similar to FIGS. 5B and 5C, but digital PCR was
performed here for BRAF in the same serial dilutions to demonstrate
mutation enrichment during the single tube reaction for the second
target, BRAF. A serial mutation dilution experiment was performed,
where KRAS and BRAF mutations were diluted at known, decreasing
amounts. The resulting mutation enrichment is inferred by the
fractional abundance detected via digital PCR (left axis). For
example, the 1:10 sample has an original mutation abundance of
about 2%, which becomes 56.4% after the single-tube reaction. This
indicates an approximate 27-fold mutation enrichment.
FIG. 7 describes multiplexed mutation enrichment directly on
genomic DNA via NaME-PrO (as described in Nucleic Acid Research
November 2016, Song et al), followed by single-tube, all-in-one
target selection, to yield sequencing-ready DNA highly enriched in
mutations.
FIG. 8 shows combined outer gene-specific primer, plus nested
gene-specific primer PCR plus `tail` primer for highly specific
target selection that includes ligated UIDs (molecular barcode) on
the other end. This can be optionally combined with NaME-PrO or
COLD-PCR for mutation enrichment. Attachment of molecular barcodes
(UID) at step 1 using ligation, is followed by an `all-in-one`
target enrichment using gene-specific primers only on one end of
each DNA target, plus the generic linker ligated in the first step.
This approach ensures that the selected DNA targets will always
contain the molecular barcode.
DETAILED DESCRIPTION
The present disclosure, in one aspect, relates to compositions and
methods for highly efficient and specific sample preparation in a
single tube to provide sequencing-ready DNA that combines (a) DNA
target selection; (b) optional incorporation of molecular barcodes
for quantification of mutation abundance, and (c) optional mutation
enrichment for increasing sequencing efficiency and reducing costs.
This is achieved by using multi-stage PCR reactions as described in
subsequent sections.
The present disclosure is based on the discovery that serial
amplification reactions can be carried out in a single vessel when
one understands the subtle relationship between temperature,
concentration, length of oligonucleotides, and number of
amplification cycles. Provided in the single reaction vessel are
(i) a sample of double-stranded DNA (e.g., genomic DNA, or cDNA);
(ii) a set of outer multiplexed primers; (iii) a set of inner
multiplexed primers; and (iv) a set of tail primers. The kinetic
conditions of the reaction can be altered to favor the annealing of
the set of outer multiplexed primers over the set of inner
multiplexed primers (and over the tail primers). For example, if
both sets of primers are the same length and present at the same
concentration, and have the same melting temperature (Tm) and
annealing temperature (Ta), the primers will anneal similarly.
However, if the set of outer multiplexed primers are present in a
10.times. excess, they will anneal more favorably than the set of
inner multiplexed primers. If the set of outer multiplexed primers
are longer than the set of inner multiplexed primers and their
annealing temperature is above the temperature that allows the set
of inner multiplexed primers to anneal, this will favor annealing
of the set of outer multiplexed primers. Once the PCR product
generated by the outer multiplexed primers starts building up, the
increased concentration of the amplified region will now offer
ample template for the inner primers to also bind substantially and
generate PCR product which is nested to the product produced by the
outer primers, and thereby being highly specific to the intended
DNA targets. Finally, the set of tail primers will anneal after the
set of outer multiplexed primers and after the set of inner
multiplexed primers because the tail sequence is not present in the
template DNA until after the amplification reaction with the inner
multiplexed primers.
If the difference in one of the following factors: annealing
temperature (which is dependent partly on the length) of primers,
concentration of primers, and amplification cycles between
different steps of any one of the methods disclosed herein, is
high, then the difference in the other factors may be lower. For
example, if the difference in the annealing temperatures for the
outer and inner primers is high (e.g., greater than 10.degree. C.),
then the difference in the concentrations of the outer primers and
inner primers may be less (e.g., less than 200 times, or less than
20 times). Similarly, if the difference in the annealing
temperatures for the tail and inner primers is high (e.g., greater
than 5, 10, 15 or 20.degree. C.), then the difference in the
concentrations of the tail primers and inner primers may be less
(e.g., less than 500 times, less than 200 times, less than 20
times, or less than 2 times). On the other hand, if the difference
in the annealing temperatures for the outer and inner primers is
low (e.g., 10.degree. C. or less), then a higher ratio of
concentrations of the outer and inner primers may be utilized
(e.g., 200 times or more, or 20 times or more). Or, if the if the
difference in the annealing temperatures for the tail and inner
primers is low (e.g., less than 20, 15, 10, 5, or 3.degree. C.),
then a higher difference in the concentrations of the tail primers
and inner primers may be utilized (e.g., 20, 200, 100, or 10,000
times or more).
As is shown in FIG. 1, the methods disclosed herein provide for
selecting and amplifying DNA targets in a single reaction vessel by
subjecting the provided contents in the single reaction vessel to
an amplification condition which favors the annealing of the set of
outer multiplexed primers to the DNA (e.g., step (b)); an
amplification condition which favors annealing of the set of inner
multiplexed primers to amplified products of step (b) (e.g., step
(c)); and an amplification condition under which the set of tail
primers anneal to the amplified products of step (c). In some
embodiments, the provided contents in the reaction vessel are
subjected to an amplification condition which favors annealing of
the set of outer multiplexed primers first, an amplification
condition which favors annealing of the set of inner multiplexed
primers second, and an amplification condition under which the set
of tail primers anneal third.
For example, in the first step of FIG. 1 (e.g., step (a)), the
contents of the reaction, including the three sets of primers and
the genomic DNA, are provided to the reaction vessel. In the second
step of FIG. 1 (e.g., step (b)), the set of outer multiplexed
primers, which are complementary to the genomic DNA, anneal to the
genomic DNA and amplify a segment of the genomic DNA. In the third
step of FIG. 1 (e.g., step (c)), the set of inner multiplexed
primers anneal to the amplified genomic DNA. The inner multiplexed
primers have a portion that is complementary to the genomic DNA and
is nested relative to the outer multiplexed primers, and have a
tail portion. A shorter segment of genomic DNA is amplified having
tail segments attached to the ends. In the fourth step of FIG. 1
(e.g., step (d)), the tail primers anneal to the tail segments on
the amplification product from step (c) and the genomic DNA portion
having tail segments at the end is further amplified. Each of these
steps will be discussed in further detail below.
In some embodiments, the sample of DNA (e.g., genomic DNA, or cDNA)
is obtained from a biological sample. The term "biological sample"
refers to any sample including tissue samples (such as tissue
sections and needle biopsies of a tissue) and cell samples (e.g.,
cytological smears (such as Pap or blood smears) or samples of
cells obtained by microdissection). Other examples of biological
samples include blood, plasma, serum, urine, semen, fecal matter,
cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus,
biopsied tissue (e.g., obtained by a surgical biopsy or needle
biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such
as buccal swabs), or any material containing biomolecules that is
derived from a first biological sample. In some embodiments, the
biological sample is fixed or frozen. In some embodiments, the
biological sample is formalin-fixed paraffin-embedded (FFPE).
As is used herein, a "reaction vessel" may be any suitable
container for subjecting the DNA (e.g., genomic DNA, or cDNA) and
primers of the claims to the amplification conditions of the
claims. In some embodiments, the reaction vessel is suitable for
subjecting DNA and primers to polymerase chain reaction (PCR). In
some embodiments, the reaction vessel comprises a tube (e.g., a
test tube, a PCR tube, or a capillary tube). In some embodiments,
the reaction vessel comprises a well of a plate (e.g., a PCR
plate).
As used herein, "primers` refers to oligonucleotides that anneal to
opposite strands of a target sequence so as to form an
amplification product during a PCR reaction.
Outer Multiplex Primers
The methods described herein for selecting and amplifying DNA
targets in a single reaction vessel, in some embodiments, comprise
subjecting the provided contents in the single reaction vessel to
an amplification condition which favors the annealing of the set of
outer multiplexed primers to the DNA (e.g., genomic DNA, or cDNA)
as shown in the second step of FIG. 1 (e.g., step (b)). In some
embodiments, the provided contents in the reaction vessel are
subjected to the amplification condition which favors annealing of
the set of outer multiplexed primers before the amplification
condition which favors annealing of the set of inner multiplexed
primers or an amplification condition under which the set of tail
primers anneal. In some embodiments, in the single-reaction tube
assay described herein, this amplification condition will favor the
annealing of the set of outer multiplexed primers to the DNA
because (i) the annealing temperature of the outer multiplex
primers is higher than the annealing temperature of the inner
multiplex primers, hence keeping the primer annealing temperature
high prevents the inner primers from binding at this higher
temperature, and (ii) because template DNA comprising the tail
sequence has not yet been generated (i.e., the tail primers do not
have template DNA to bind to). In addition, the outer multiplex
primers can be present at a higher concentration than the inner
multiplex primers, further favoring the annealing of the outer
multiplex primers at temperatures above the annealing temperature
of the inner multiplex primers. In some embodiments, in the
single-reaction tube assay described herein, this amplification
condition will favor the annealing of the set of outer multiplexed
primers to the DNA because (i) the outer multiplex primers are
present at a higher concentration than the inner multiplex primers,
(ii) the annealing temperature of the outer multiplex primers is
lower than the annealing temperature of the inner multiplex
primers, but the higher concentration of the outer primers favors
outer primer annealing at or below the outer primer annealing
temperature, and (iii) because template DNA comprising the tail
sequence has not yet been generated (i.e., the tail primers do not
have template DNA to bind to).
In some embodiments, the annealing temperature (T.sub.a) of the set
of outer multiplex primers is about 45.degree. C., 46.degree. C.,
47.degree. C., 48.degree. C., 49.degree. C., 50.degree. C.,
51.degree. C., 52.degree. C., 53.degree. C., 54.degree. C.,
55.degree. C., 56.degree. C., 57.degree. C., 58.degree. C.,
59.degree. C., 60.degree. C., 61.degree. C., 62.degree. C.,
63.degree. C., 64.degree. C., 65.degree. C., 66.degree. C.,
67.degree. C., 68.degree. C., 69.degree. C., or 70.degree. C. In
some embodiments, the T.sub.a of the set of outer multiplex primers
is about 55.degree. C. to about 70.degree. C. In some embodiments,
the T.sub.a of the set of outer multiplex primers is about
60.degree. C. to about 65.degree. C. In some embodiments, the
T.sub.a of the set of outer multiplex primers is about 58.degree.
C. to about 62.degree. C.
In some embodiments, the contents in the single reaction vessel are
subjected to amplification at the annealing temperature of outer
multiplex primers for 6, 7, 8, 9, 10, 11, 12, 13, or 14 cycles. In
some embodiments, the contents in the single reaction vessel are
subjected to amplification at the annealing temperature of the set
of outer multiplex primers for more than 14 cycles. In some
embodiments, the contents in the single reaction vessel are
subjected to amplification at the annealing temperature of the set
of outer multiplex primers for 6-14 cycles, or 8-12 cycles.
In some embodiments, the outer multiplex primers comprise an outer
forward primer and an outer reverse primer. In some embodiments,
the outer forward and reverse primers complement target nucleic
acids on the DNA (e.g., genomic DNA, or cDNA).
In some embodiments, either the outer forward primer or the outer
reverse primer complements a common tag and the other primer
complements target nucleic acids on the DNA (e.g., genomic DNA, or
cDNA).
In some embodiments, the outer forward primer is 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, or 50 nucleotides in length. In some embodiments, the outer
forward primer is greater than 40 nucleotides in length. In some
embodiments, the outer forward primer is about 10 to 40
nucleotides, or about 15-35 nucleotides in length.
In some embodiments, the outer reverse primer is 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, or 50 nucleotides in length. In some embodiments, the outer
reverse primer is greater than 40 nucleotides in length. In some
embodiments, the outer reverse primer is about 10 to 40
nucleotides, or about 15-35 nucleotides in length.
The concentration of the outer primers (forward and reverse outer
primers) is selected to be lower than, or in the range of primer
concentrations that are typically used in PCR. It is selected so
that the outer primers make very little product compared to the
inner and tail primers. This is to avoid amplification of
mis-primed targets, especially in samples of genomic DNA, where the
likelihood of mis-priming is particularly high.
In some embodiments, the concentration of the outer forward primer
is 0.005 to 0.4 .mu.M. In some embodiments, the concentration of
the outer forward primer is 0.01 to 0.2 .mu.M. In some embodiments,
the concentration of the outer forward primer is 0.005, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13,
0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.30, or 0.40 .mu.m.
In some embodiments, the concentration of the outer reverse primer
is 0.005 to 0.4 .mu.M. In some embodiments, the concentration of
the outer reverse primer is 0.01 to 0.2 .mu.M. In some embodiments,
the concentration of the outer reverse primer is 0.005, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13,
0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.30, or 0.40 .mu.m.
In some embodiments, provided in the single reaction vessel are two
or more sets of outer multiplexed primers that are complementary to
two or more different targets. In some embodiment, the at least two
sets of outer multiplexed primers is at least 5, 10, 15, 20, 30,
40, 50, 100, 200, 500, 1,000, 5,000, 10,000 or 30,000 outer
multiplexed primers.
Inner Multiplex Primers
The methods described herein for selecting and amplifying DNA
targets in a single reaction vessel, in some embodiments, comprise
subjecting the provided contents in the single reaction vessel to a
second amplification condition which favors the annealing of the
set of inner multiplexed primers to amplified products of the outer
multiplexed primers as shown in the third step of FIG. 1 (e.g.,
step (c)). In some embodiments, the provided contents in the
reaction vessel are subjected to the second amplification condition
which favors annealing of the set of inner multiplexed primers
after the amplification condition which favors annealing of the set
of outer multiplexed primers and before an amplification condition
under which the set of tail primers anneal. In some embodiments, in
the single-reaction tube assay described herein, the inner
multiplex primers are at a concentration that is lower than the
concentration of the outer multiplex primers but the annealing
temperature of the inner multiplex primers is above the annealing
temperature of the outer multiplex primers. The second
amplification condition will favor the annealing of the set of
inner multiplexed primers to the product amplified by the outer
multiplex primers, for example, if the annealing is carried out at
a temperature above the annealing temperature of the outer
multiplex primers (and above the annealing temperature of the tail
primers). In embodiments of the single-reaction tube assay
described herein, the second amplification condition will favor the
annealing of the set of inner multiplexed primers versus the outer
multiplex primers and the tail primers. Amplification by the set of
tail primers can predominantly occur third, in part, because the
tail primers amplify only the amplification product of the inner
multiplexed primers.
In some embodiments, the annealing temperature (T.sub.a) of the set
of inner multiplex primers is about 45.degree. C., 46.degree. C.,
47.degree. C., 48.degree. C., 49.degree. C., 50.degree. C.,
51.degree. C., 52.degree. C., 53.degree. C., 54.degree. C.,
55.degree. C., 56.degree. C., 57.degree. C., 58.degree. C.,
59.degree. C., 60.degree. C., 61.degree. C., 62.degree. C.,
63.degree. C., 64.degree. C., 65.degree. C., 66.degree. C.,
67.degree. C., 68.degree. C., 69.degree. C., or 70.degree. C. In
some embodiments, the T.sub.a of the set of inner multiplex primers
is about 45.degree. C. to about 60.degree. C., or about 50.degree.
C. to about 55.degree. C. In some embodiments, the T.sub.a of the
set of inner multiplex primers is about 66.degree. C. to about
70.degree. C.
In some embodiments, the annealing temperatures of the inner
multiplexed primers is different from annealing temperature of the
outer multiplexed primers. In some embodiments, the annealing
temperatures of the inner multiplexed primers is 1.degree. C.,
2.degree. C., 3.degree. C., 4.degree. C., 5.degree. C., 6.degree.
C., 7.degree. C., 8.degree. C., 9.degree. C., 10.degree. C.,
11.degree. C., 12.degree. C., 13.degree. C., 14.degree. C.,
15.degree. C., 16.degree. C., 17.degree. C., 18.degree. C.,
19.degree. C., 20.degree. C., 21.degree. C., 22.degree. C.,
23.degree. C., 24.degree. C., 25.degree. C., 26.degree. C.,
27.degree. C., 28.degree. C., 29.degree. C., 30.degree. C.,
31.degree. C., 32.degree. C., 33.degree. C., 34.degree. C.,
35.degree. C., 36.degree. C., 37.degree. C., 38.degree. C.,
39.degree. C., 40.degree. C., 41.degree. C., 42.degree. C.,
43.degree. C., 44.degree. C., 45.degree. C., 46.degree. C.,
47.degree. C., 48.degree. C., 49.degree. C. or 50.degree. C.
different from annealing temperature of the outer multiplexed
primers. In some embodiments, the annealing temperatures of the
inner multiplexed primers is 1.degree. C.-50.degree. C. different
from annealing temperature of the outer multiplexed primers. In
some embodiments, the annealing temperatures of the inner
multiplexed primers is 3.degree. C. -20.degree. C. different from
annealing temperature of the outer multiplexed primers.
In some embodiments, the annealing temperatures of the inner
multiplexed primers is lower than the annealing temperature of the
outer multiplexed primers. In some embodiments, the annealing
temperatures of the inner multiplexed primers is 1.degree. C.,
2.degree. C., 3.degree. C., 4.degree. C., 5.degree. C., 6.degree.
C., 7.degree. C., 8.degree. C., 9.degree. C., 10.degree. C.,
11.degree. C., 12.degree. C., 13.degree. C., 14.degree. C.,
15.degree. C., 16.degree. C., 17.degree. C., 18.degree. C.,
19.degree. C., 20.degree. C., 21.degree. C., 22.degree. C.,
23.degree. C., 24.degree. C., 25.degree. C., 26.degree. C.,
27.degree. C., 28.degree. C., 29.degree. C., 30.degree. C.,
31.degree. C., 32.degree. C., 33.degree. C., 34.degree. C.,
35.degree. C., 36.degree. C., 37.degree. C., 38.degree. C.,
39.degree. C., 40.degree. C., 41.degree. C., 42.degree. C.,
43.degree. C., 44.degree. C., 45.degree. C., 46.degree. C.,
47.degree. C., 48.degree. C., 49.degree. C. or 50.degree. C. lower
than the annealing temperature of the outer multiplexed primers. In
some embodiments, the annealing temperatures of the inner
multiplexed primers is 1.degree. C.-50.degree. C. lower than the
annealing temperature of the outer multiplexed primers. In some
embodiments, the annealing temperatures of the inner multiplexed
primers is 3.degree. C. -20.degree. C. lower than the annealing
temperature of the outer multiplexed primers. In some embodiments,
the annealing temperature of the outer multiplexed primers is
60-65.degree. C., and the annealing temperature of the inner
multiplexed primers is 50-55.degree. C.
In some embodiments, the annealing temperatures of the inner
multiplexed primers is higher than the annealing temperature of the
outer multiplexed primers. In some embodiments, the annealing
temperatures of the inner multiplexed primers is 1.degree. C.,
2.degree. C., 3.degree. C., 4.degree. C., 5.degree. C., 6.degree.
C., 7.degree. C., 8.degree. C., 9.degree. C., 10.degree. C.,
11.degree. C., 12.degree. C., 13.degree. C., 14.degree. C.,
15.degree. C., 16.degree. C., 17.degree. C., 18.degree. C.,
19.degree. C., 20.degree. C., 21.degree. C., 22.degree. C.,
23.degree. C., 24.degree. C., 25.degree. C., 26.degree. C.,
27.degree. C., 28.degree. C., 29.degree. C., 30.degree. C.,
31.degree. C., 32.degree. C., 33.degree. C., 34.degree. C.,
35.degree. C., 36.degree. C., 37.degree. C., 38.degree. C.,
39.degree. C., 40.degree. C., 41.degree. C., 42.degree. C.,
43.degree. C., 44.degree. C., 45.degree. C., 46.degree. C.,
47.degree. C., 48.degree. C., 49.degree. C. or 50.degree. C. higher
than the annealing temperature of the outer multiplexed primers. In
some embodiments, the annealing temperatures of the inner
multiplexed primers is 1.degree. C.-50.degree. C. higher than the
annealing temperature of the outer multiplexed primers. In some
embodiments, the annealing temperatures of the inner multiplexed
primers is 3.degree. C.-20.degree. C. higher than the annealing
temperature of the outer multiplexed primers. In some embodiments,
the annealing temperature of the outer multiplexed primers is
58-62.degree. C., and the annealing temperature of the inner
multiplexed primers is 66-70.degree. C.
In some embodiments, the contents in the single reaction vessel are
subjected to amplification at the annealing temperature of the set
of inner multiplex primers for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
cycles. In some embodiments, the contents in the single reaction
vessel are subjected to amplification at the annealing temperature
of the set of inner multiplex primers for 2-8 cycles, or 2-6
cycles.
In some embodiments, the number of amplification cycles in the
amplification condition which favors the annealing of the set of
outer multiplexed primers to the DNA (e.g., step (b)) exceeds the
number of amplification cycles in the amplification condition which
favors the annealing of the set of inner multiplexed primers to
amplified products of the outer multiplexed primers (e.g., step
(c)). In some embodiments, step (b) exceeds step (c) by 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 cycles.
In some embodiments, the inner multiplex primers comprise an inner
forward primer and an inner reverse primer. In some embodiments,
each of the inner forward and reverse primers comprises a
target-specific anchor on its 3' end (e.g., an forward and reverse
target-specific anchor), and the inner forward primer comprises a
common forward tail on its 5' end and the inner reverse primer
comprises a common reverse tail on its 5' end. In some embodiments,
the target-specific anchor of the inner forward primer is 3' to the
outer forward primer. In some embodiments, the target-specific
anchor of the inner reverse primer is 5' to the outer reverse
primer.
In some embodiments, (ii) the inner forward primer is complementary
to the common tag, which comprises a common forward tail, and
wherein the inner reverse primer comprises a target-specific anchor
on its 3' end and a common reverse tail on its 5' end, or (ii) the
inner reverse primer is complementary to the common tag, which
comprises a common reverse tail, and wherein the inner forward
primer comprises a target-specific anchor on its 3' end and a
common forward tail on its 5' end.
In some embodiments, the common forward tail is different from the
common reverse tail.
In some embodiments, the forward target-specific anchor is 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, or 50 nucleotides in length. In some embodiments,
the forward target-specific anchor is greater than 40 nucleotides
in length. In some embodiments, the forward target-specific anchor
is about 10 to 40 nucleotides, or about 15-35 nucleotides in
length.
In some embodiments, the reverse target-specific anchor is 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, or 50 nucleotides in length. In some embodiments,
the reverse target-specific anchor is greater than 40 nucleotides
in length. In some embodiments, the reverse target-specific anchor
is about 10 to 40 nucleotides, or about 15-35 nucleotides in
length.
In some embodiments, the common forward tail comprises sequence
that is common to all of the inner forward primers. In some
embodiments, the common reverse tail comprises sequence that is
common to all of the inner reverse primers. In some embodiments the
common forward tail and the common reverse tail comprise sequencing
primer sequence. In some embodiments, the tail primers comprise 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, or 35 base pairs, e.g., 20-30 base pairs of a 3'end portion
of a sequencing adapter. In some embodiments the common forward
tail and the common reverse tail comprise NGS adaptor sequence. In
some embodiments, the common forward tail and the common reverse
tail comprise Illumina.RTM. sequencing adaptor sequence. In some
embodiments, the common forward tail and the common reverse tail
comprise Qiagen.RTM. sequencing adaptor sequence. In some
embodiments, the common forward tail and the common reverse tail
comprise Ion Torrent.RTM. sequencing adaptor sequence.
In some embodiments, the common forward tail is 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or 50 nucleotides in length. In some embodiments, the common
forward tail is greater than 40 nucleotides in length. In some
embodiments, the common forward tail is about 10 to 40 nucleotides,
or about 15-35 nucleotides, or about 20-30 nucleotides in
length.
In some embodiments, the common reverse tail is 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or 50 nucleotides in length. In some embodiments, the common
reverse tail is greater than 40 nucleotides in length. In some
embodiments, the common reverse tail is about 10 to 40 nucleotides,
or about 15-35 nucleotides, or about 20-30 nucleotides in
length.
In some embodiments, the concentration of the inner primers
(forward and reverse inner primers) is selected to be much lower
than the concentration of outer primers, such that if the outer
primers were absent, or if the first step of amplification of any
one of the methods described herein were not performed, then
amplification using the inner primers would yield only an
insignificant amount of product from the DNA sample (e.g., about
100 times less, 1000 times less, 10,000 times less, or 100,000
times less). In some embodiments, the concentration of the inner
forward primer is 0.0005 to 0.08 .mu.M. In some embodiments, the
concentration of the inner forward primer is 0.001 to 0.04 .mu.M.
In some embodiments, the concentration of the inner forward primer
is 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,
0.009, 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017,
0.018, 0.019, 0.020, 0.030, 0.040, 0.050, 0.060, 0.070, or 0.080
.mu.M.
In some embodiments, the concentration of the inner reverse primer
is 0.0005 to 0.08 .mu.M. In some embodiments, the concentration of
the inner reverse primer is 0.001 to 0.04 .mu.M. In some
embodiments, the concentration of the inner reverse primer is
0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,
0.009, 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017,
0.018, 0.019, 0.020, 0.030, 0.040, 0.050, 0.060, 0.070, or 0.080
.mu.M.
As detailed in the Summary section above and as is made clear in
FIG. 1, 3, or 7, in some embodiments, the ratio of concentration of
the inner multiplexed primers to the concentration of outer
multiplexed primers is 2:10, 2:20, 2:30, 2:40, 2:50, 2:60, 2:70,
2:80, 2:90, 2:100, 2:120, 2:140, 2:160, 2:180, 2:200, 2:220, 2:240,
2:260, 2:280, 2:300, 2:320, 2:340, 2:360, 2:380, 2:400, 2:450,
2:500, 2:550, 2:600, 2700, 2:800, 2:900, 2:1000, 2:1100, 2:1200,
2:1300, 2:1400, 2:1500, 2:1600, 2:1700, 2:1800, 2:1900, or 2:2000.
In some embodiments, the ratio of concentration of the outer
multiplexed primers to the concentration of inner multiplexed
primers is 2:20-2:2000. In some embodiments, the ratio of
concentration of the outer multiplexed primers to the concentration
of inner multiplexed primers is 2:100-2:300.
In some embodiments, the concentration of outer primers compared to
the concentration of inner primers is significantly high so that if
the outer primers were absent, the inner primers would not make a
significant amount of product from the DNA sample as a template. In
some embodiments, the ratio of concentration of the outer
multiplexed primers to the concentration of inner multiplexed
primers is 0.25-2000 (e.g., 0.25-2000, 0.25-200, 0.25-50, 0.25-20,
0.25-2, 2-2000, 1-2000, 1-200, 1-50, 1-20, 1-2, 2-2000, 2-200, or
2-20). For example, the concentration of outer multiplexed primers
may be 0.01-0.2 .mu.M (e.g., 0.01-0.2, or 0.02-0.1 .mu.M) and the
concentration of inner multiplexed primers may be 0.0001-0.04 .mu.M
(e.g., 0.0001-0.04, or 0.001-0.01 .mu.M)
In some embodiments, provided in the single reaction vessel are two
or more sets of inner multiplexed primers that are complementary to
two or more different targets. In some embodiment, the at least two
sets of outer multiplexed primers is at least 5, 10, 15, 20, 30,
40, 50, 100, 200, 500, 1,000, 5,000, 10,000 or 30,000 outer
multiplexed primers.
Tail Primers
The methods described herein for selecting and amplifying DNA
targets in a single reaction vessel, in some embodiments, comprise
subjecting the provided contents in the single reaction vessel to
an amplification condition under which the set of tail primers
anneal to the amplified products of the inner multiplexed primers
as shown in the fourth step of FIG. 1 (e.g., step (d)). In some
embodiments, the provided contents in the reaction vessel are
subjected to the amplification condition under which the set of
tail primers anneal after the amplification condition which favors
annealing of the set of outer multiplexed primers and after the
amplification condition which favors annealing of the set of inner
multiplexed primers. In some embodiments, in the single-reaction
tube assay described herein, the tail primers will anneal in this
amplification condition because (i) the outer multiplexed primers
will predominantly amplify first because the outer multiplex
primers are present at a higher concentration than the inner
multiplex primers, (ii) the inner multiplexed primers will then
predominantly amplify second, and (iii) amplification by the set of
tail primers will predominantly occur third, because the tail
primers amplify the amplification product of the inner multiplexed
primers. In some embodiments, the tail primers anneal only at a
temperature below the annealing temperature of the outer and inner
multiplex primers. In these embodiments, the tail primers are
typically at a higher concentration than the outer and inner
multiplex primers, such that tail primer annealing will be favored
when the temperature is at or below the annealing temperature of
the tail primer.
In some embodiments, the annealing temperature (T.sub.a) of the set
of tail primers is about 45.degree. C., 46.degree. C., 47.degree.
C., 48.degree. C., 49.degree. C., 50.degree. C., 51.degree. C.,
52.degree. C., 53.degree. C., 54.degree. C., 55.degree. C.,
56.degree. C., 57.degree. C., 58.degree. C., 59.degree. C.,
60.degree. C., 61.degree. C., 62.degree. C., 63.degree. C.,
64.degree. C., 65.degree. C., 66.degree. C., 67.degree. C.,
68.degree. C., 69.degree. C., or 70.degree. C. In some embodiments,
the T.sub.a of the set of tail primers is about 55.degree. C. to
about 70.degree. C., or about 60.degree. C. to about 70.degree.
C.
In some embodiments, the annealing temperatures of the tail primers
is different from annealing temperature of the inner multiplexed
primers. In some embodiments, the annealing temperatures of the
tail primers is 1.degree. C., 2.degree. C., 3.degree. C., 4.degree.
C., 5.degree. C., 6.degree. C., 7.degree. C., 8.degree. C.,
9.degree. C., 10.degree. C., 11.degree. C., 12.degree. C.,
13.degree. C., 14.degree. C., 15.degree. C., 16.degree. C.,
17.degree. C., 18.degree. C., 19.degree. C., 20.degree. C.,
21.degree. C., 22.degree. C., 23.degree. C., 24.degree. C.,
25.degree. C., 26.degree. C., 27.degree. C., 28.degree. C.,
29.degree. C., 30.degree. C., 31.degree. C., 32.degree. C.,
33.degree. C., 34.degree. C., 35.degree. C., 36.degree. C.,
37.degree. C., 38.degree. C., 39.degree. C., 40.degree. C.,
41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C. or 50.degree. C. different from annealing temperature
of the inner multiplexed primers. In some embodiments, the
annealing temperatures of the tail primers is 1.degree.
C.-50.degree. C. different from annealing temperature of the inner
multiplexed primers. In some embodiments, the annealing
temperatures of the tail primers is 3.degree. C. -20.degree. C.
different from annealing temperature of the inner multiplexed
primers.
In some embodiments, the contents in the single reaction vessel are
subjected to amplification at the annealing temperature of the set
of tail primers for 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30 cycles. In some embodiments, the
contents in the single reaction vessel are subjected to
amplification at the annealing temperature of the set of tail
primers for more than 30 cycles. In some embodiments, the contents
in the single reaction vessel are subjected to amplification at the
annealing temperature of the set of tail primers for 10-30
cycles.
In some embodiments, the number of amplification cycles in an
amplification condition under which the set of tail primers anneal
to the amplified products of the inner multiplexed primers (e.g.,
step (d)) exceeds the number of amplification cycles in the
amplification condition which favors the annealing of the set of
inner multiplexed primers to amplified products of the outer
multiplexed primers (e.g., step (c)). In some embodiments, step (d)
exceeds step (c) by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more
cycles.
In some embodiments, the number of amplification cycles in an
amplification condition under which the set of tail primers anneal
to the amplified products of the inner multiplexed primers (e.g.,
step (d)) exceeds the number of amplification cycles in the
amplification condition which favors the annealing of the set of
outer multiplexed primers to the DNA (e.g., step (b)). In some
embodiments, step (d) exceeds step (b) by 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more cycles.
In some embodiments, the set of tail primers comprises a first tail
primer and a second tail primer. In some embodiments, the first
tail primer is complementary to the common forward tail and the
second tail primer is complementary to the common reverse tail.
In some embodiments, the first tail primer is 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or 50 nucleotides in length. In some embodiments, the first
tail primer is greater than 40 nucleotides in length. In some
embodiments, the first tail primer is about 10 to 40 nucleotides,
or about 15-35 nucleotides, or about 20-30 nucleotides in
length.
In some embodiments, the second tail primer is 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or 50 nucleotides in length. In some embodiments, the second
tail primer is greater than 40 nucleotides in length. In some
embodiments, the second tail primer is about 10 to 40 nucleotides,
or about 15-35 nucleotides, or about 20-30 nucleotides in
length.
The concentration of tail primers (forward and reverse tail
primers) is high compared to the concentration of inner primers,
and sometimes compared to the outer primers as well, such that the
tail primers make the most product compared to the outer and inner
primers. In some embodiments, the concentration of the first tail
primer is 0.01 to 1.0 .mu.M. In some embodiments, the concentration
of the first tail primer is 0.05 to 0.5 .mu.M. In some embodiments,
the concentration of the first tail primer is 0.01, 0.05, 0.10,
0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, or 1.0 .mu.M.
In some embodiments, the concentration of the second tail primer is
0.01 to 1.0 .mu.M. In some embodiments, the concentration of the
second tail primer is 0.05 to 0.5 .mu.M. In some embodiments, the
concentration of the second tail primer is 0.01, 0.05, 0.10, 0.15,
0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, or 1.0 .mu.M.
As detailed in the Summary section above and as is made clear in
FIG. 1, 3, or 7, in some embodiments, the ratio of concentration of
the inner primers to the concentration of tail multiplexed primers
is 2.5:100, 2.5:200, 2.5:300, 2.5:400, 2.5:500, 2.5:600, 2.5:700,
2.5:800, 2.5:900, 2.5:1000, 2.5:1100, 2.5:1200, 2.5:1300, 2.5:1400,
2.5:1500, 2.5:1600, 2.5:1700, 2.5:1800, 2.5:1900, 2.5:2000,
2.5:2500, 2.5:3000, 2.5:4000, 2.5:4500, or 2.5:5000. In some
embodiments, the ratio of concentration of the tail primers to the
concentration of inner multiplexed primers is 2.5:100-2.5-5:000. In
some embodiments, the ratio of concentration of the tail primers to
the concentration of inner multiplexed primers is
2.5:500-2.5:1500.
In some embodiments, the ratio of concentration of the tail primers
to the concentration of inner multiplexed primers is 2.5-10,000
(e.g., 2.5-10,000, 5-10,000, 5-1,000, 5-200, 5:100, 5-10, 10-100,
10-1,000, 100-1,000, or 1,000-10,000). For example, the
concentration of tail primers may be 0.1-1 .mu.M (e.g., 0.1-0.5,
0.1-0.2, 0.2-1, 0.3-1, 0.4-0.8, or 0.5-1 .mu.M) and the
concentration of inner multiplexed primers may be 0.0001-0.01 .mu.M
(e.g., 0.0001-0.01, or 0.001-0.01 .mu.M).
As detailed in the Summary section above and as is made clear in
FIG. 1, 3, or 7, in some embodiments, the ratio of the
concentration of the outer primer to the concentration of the inner
multiplexed primers is 0.5:10, 0.5:20, 0.5:30, 0.5:40, 0.5:50,
0.5:60, 0.5:70, 0.5:80, 0.5:90, 0.5:100, 0.5:120, 0.5:140, 0.5:160,
0.5:180, 0.5:200, 0.5:220, 0.5:240, 0.5:260, 0.5:280, 0.5:300,
0.5:320, 0.5:340, 0.5:360, 0.5:380, 0.5:400, 0.5:450, 0.5:500,
0.5:550, 0.5:600, 0.5:700, 0.5:800, 0.5:900, or 0.5:1000. In some
embodiments, the ratio of the concentration of the tail primer to
the concentration of the outer multiplexed primers is
0.5:10-0.5:1000. In some embodiments, the ratio of the
concentration of the tail primer to the concentration of the outer
multiplexed primers is 0.5:70-0.5:150.
In some embodiments, the ratio of concentration of the tail primers
to the concentration of outer multiplexed primers is 0.5-200 (e.g.,
0.5-200, 0.5-50, 1-20, 5-200, 5-100, 5-50, 10-200, 10-50, or
50-200). For example, the concentration of tail primers may be
0.1-1 .mu.M (e.g., 0.1-0.5, 0.1-0.2, 0.2-1, 0.3-1, 0.4-0.8, or
0.5-1 .mu.M) and the concentration of outer multiplexed primers may
be 0.01-0.2 .mu.M (e.g., 0.01-0.2, or 0.02-0.1 .mu.M).
Hot Start Primers
In some embodiments, a pair of primer (e.g., outer primers, inner
primers, or tail primers) is a pair of hot start primers. Hot start
primers contain a thermolabile chemical modification that allows
hot start activation in PCR, for example, in some embodiments, the
primers may have a 4-oxo-tetradecyl (OXT) phosphotriester groups
introduced at the 3'-terminal phosphodiester linkages.
In some embodiments, the set of inner multiplexed primers are hot
start primers and the set of inner multiplexed primers are
activated by subjecting the provided contents in the single
reaction vessel to an activation temperature after subjecting the
provided contents in the single reaction vessel to an amplification
condition which favors the annealing of the set of outer
multiplexed primers to the DNA (e.g., after step (b)). In some
embodiments, the set of tail primers are hot start primers and the
set of tail primers are activated by subjecting the provided
contents in the single reaction vessel to an activation temperature
after subjecting the provided contents in the single reaction
vessel to an amplification condition which favors annealing of the
set of inner multiplexed primers (e.g., after step (c)). In some
embodiments, the amplification reaction further comprises primers
for sequencing adaptor addition and the primers for sequencing
adaptor addition are hot start primers.
In some embodiments, the activation temperature of the hot start
primers is 85.degree. C., 86.degree. C., 87.degree. C., 88.degree.
C., 89.degree. C., 90.degree. C., 91.degree. C., 92.degree. C.,
93.degree. C., 94.degree. C., 95.degree. C., 96.degree. C.,
97.degree. C., 98.degree. C., 99.degree. C., 100.degree. C., or
greater, e.g., 90.degree. C.-95.degree. C.
In some embodiments, the hot start primers are activated after
being subjected to an activation temperature for 1, 2, 3, 4, 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, or 55 seconds, or 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, or 15 or more minutes. In some
embodiments, the hot start primers are activated after being
subjected to an activation temperature for 5 seconds to 5 minutes.
In some embodiments, the hot start primers are activated after
being subjected to an activation temperature for 2 minutes to 10
minutes.
PCR
In some embodiments, amplification can be performed using a
polymerase chain reaction (PCR). As used herein, PCR can refer to a
reaction for the in vitro amplification of specific DNA sequences
by the simultaneous primer extension of complementary strands of
DNA.
The temperature of the reaction solutions may be sequentially
cycled between a denaturing state, an annealing state, and an
extension state for a predetermined number of cycles. The actual
times and temperatures can be enzyme, primer, and target
dependent.
For any given reaction, denaturing states can range in certain
embodiments from about 75.degree. C. to about 100.degree. C. The
annealing temperature and time can influence the specificity and
efficiency of primer binding to a particular locus within a target
nucleic acid and may be important for particular PCR reactions.
As is described herein, for any given reaction, annealing states
can range in certain embodiments from about 20.degree. C. to about
75.degree. C.
Extension temperature and time may impact the allele product yield
and are understood to be an inherent property of the enzyme under
study. For a given enzyme, extension states can range in certain
embodiments from about 60.degree. C. to about 75.degree. C.
In any of the foregoing embodiments, any DNA or RNA polymerase
(enzyme that catalyzes polymerization of nucleotides into a nucleic
acid strand) may be utilized, including thermostable polymerases
and reverse transcriptases (RTases). Examples include Bacillus
stearothermophilus pol I, Thermus aquaticus (Taq) pol I, Pyrccoccus
furiosus (Pfu), Pyrococcus woesei (Pwo), Thermus flavus (Tfl),
Thermus thermophilus (Tth), Thermus litoris (Tli) and Thermotoga
maritime (Tma). These enzymes, modified versions of these enzymes,
and combination of enzymes, are commercially available from vendors
including Roche, Invitrogen, Qiagen, Stratagene, and Applied
Biosystems. Representative enzymes include PHUSION.RTM. (New
England Biolabs, Ipswich, Mass.), Hot MasterTaq.TM. (Eppendorf),
PHUSION.RTM. Mpx (Finnzymes), PyroStart.RTM. (Fermentas), KOD (EMD
Biosciences), Z-Taq (TAKARA), and CS3AC/LA (KlenTaq, University
City, Mo.).
Salts and amplification buffers include those familiar to those
skilled in the art, including those comprising MgCl.sub.2, and
Tris-HCl and KCl, respectively. Amplification buffers may contain
additives such as surfactants, dimethyl sulfoxide (DMSO), glycerol,
bovine serum albumin (BSA) and polyethylene glycol (PEG), as well
as others familiar to those skilled in the art. Nucleotides are
generally deoxyribonucleoside triphosphates, such as deoxyadenosine
triphosphate (dATP), deoxycytidine triphosphate (dCTP),
deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate
(dTTP), and are also added to a reaction adequate amount for
amplification of the target nucleic acid.
Mutant Enrichment
Mutation enrichment technologies, e.g., COLD-PCR and NaME-PrO,
enhance mutation-containing DNA of rare alleles prior to
sequencing, thus enabling rapid and efficient sequencing where the
result can be obtained with very few sequence reads.
In some embodiments, the methods described herein comprise
enriching mutant alleles of the target nucleic acids relative to
wild-type alleles of the target nucleic acids. In some embodiments,
the methods comprise enriching mutant alleles of the target nucleic
acids relative to wild-type alleles of the target nucleic acids
before subjecting the provided contents in the single reaction
vessel to an amplification reactions described herein. In some
embodiments, the methods comprise enriching mutant alleles of the
target nucleic acids relative to wild-type alleles of the target
nucleic acids after subjecting the provided contents in the single
reaction vessel to an amplification condition under which the set
of tail primers anneal (e.g., after step (d)). In some embodiments,
the methods comprise enriching mutant alleles of the target nucleic
acids relative to wild-type alleles of the target nucleic acids
while subjecting the provided contents in the single reaction
vessel to an amplification condition under which the set of tail
primers anneal (e.g., during, or nested within step (d)).
Several methods of enriching mutant target sequence relative to
wild-type target sequence are known in the art. Non-limiting
examples of mutation enrichment methods include Nuclease-assisted
Minor-allele Enrichment using Probe Overlap (NaME-PrO),
Coamplification at Lower Denaturation temperature-PCR (COLD-PCR),
Improved and Complete Enrichment COLD-PCR (ice-COLD-PCR),
Temperature-Tolerant ice-COLD-PCR (TT-ice-COLD-PCR), toehold PCR,
and Differential Strand Separation at Critical Temperature
(DiSSECT).
NaMe or NaMe-PrO methods are described in PCT/US2016/039167, which
is incorporated by reference in its entirety. Use of NaMe-PrO in
the methods described herein is shown in FIG. 7. NaMe-PrO is shown
as the first step, before the amplification methods described
herein. A non-limiting example of a NaMe-PrO protocol includes:
(a) preparing an amplification reaction mixture comprising the
double-stranded mutant and wild-type target nucleic acids, a
thermostable double strand-specific nuclease (DSN), PCR
amplification components, and a pair of oligonucleotide probes, one
of which is complementary to the wild-type nucleic acid top strand
and the other is complementary to the wild-type nucleic acid bottom
strand, wherein the probes may overlap each other by 10-15 probes
such that the overlap coincides with the target region or be
non-overlapping and contiguous;
(b) subjecting the reaction mixture to a denaturing temperature to
permit denaturation of the wild-type nucleic acid and the mutant
target nucleic acid;
(c) reducing the temperature to permit hybridization of the probes
to their corresponding sequences on the wild-type and mutant target
nucleic acids thereby forming complementary wild-type-probe
duplexes, wherein the DSN cleaves the complementary wild-type-probe
duplexes but not the partially complementary target mutant-probe
duplexes; and
(d) subjecting the reaction mixture to an amplification condition
thereby enriching the uncleaved mutant target nucleic acid relative
to the cleaved wild-type nucleic acid.
In some embodiments, an overlap of NaMe-PrO probes coincides with
one or more mutations. In some embodiments, NaMe-PrO probes have a
3'-terminal polymerase block. In some embodiments, the probes are
complementary to SNPs near target mutations.
In some embodiments, NaMe-PrO is performed before the amplification
reactions described herein, e.g., before providing the
double-stranded DNA (e.g., genomic DNA, or cDNA), the set of outer
multiplexed primers, the set of inner multiplexed primers and the
set of tail primers in the single reaction vessel. In some
embodiments, NaMe-PrO is performed after the amplification
reactions described herein.
In some embodiments, NaMe or NaMe-PrO is then followed by
amplification of remaining mutant and wild-type target nucleic
acids according to the methods described herein.
In some embodiments, NaMe-PrO with or without amplification results
in mutation enrichment relative to wild-type target nucleic acids
of 1-200-fold (e.g., 1-150-, 5-100- or 10-100-fold) compared to the
unenriched sample. In some embodiments, NaMe-PrO with or without
amplification results in mutation enrichment relative to wild-type
target nucleic acids of more than 200-fold (e.g., 250-fold or
300-fold).
In some embodiments, a form of COLD-PCR (e.g., ice-COLD-PCR,
TT-ice-COLD-PCR or oscillating COLD-PCR) is used to enrich mutant
target nucleic acids relative to wild-type target nucleic acids.
Methods of COLD-PCR and oscillating COLD-PCR are described in WO
2009/017784, which is incorporated by reference herein in its
entirety. Use of COLD-PCR in the methods described herein is shown
at least in FIG. 3.
In the first step of FIG. 3 (e.g., step (a)), the contents of the
reaction, including the three sets of primers, the genomic DNA, and
the COLD-PCR reference sequence are provided to the reaction
vessel. In the second step of FIG. 3 (e.g., step (b)), the set of
outer multiplexed primers, which are complementary to the genomic
DNA, anneal to the genomic DNA and amplify a segment of the genomic
DNA. In the third step of FIG. 3 (e.g., step (c)), the set of inner
multiplexed primers anneal to the amplified genomic DNA and a
shorter segment of genomic DNA is amplified having tail segments
attached to the ends. In the fourth step of FIG. 3 (e.g., step
(d)), the tail primers anneal to the tail segments on the
amplification product from step (c) and the genomic DNA portion
having tail segments at the end is further amplified. COLD-PCR
occurs during the fourth step of FIG. 3, with the annealing
temperature being the critical temperature for COLD-PCR (as
described below). The reference sequence, which is perfectly
complementary to the wild type sequence, binds to the wild type
sequence but not the mutant sequence, allowing preferential
amplification of the mutant sequence. The concentration of the
reference is kept low, e.g., 1-10 nM, so that it does not
appreciably bind the amplification product in the first
amplification step.
In some embodiments, the concentration of the reference sequence is
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nM.
A non-limiting example of a COLD-PCR protocol includes:
(a) denaturing the double-stranded mutant and wild-type target
nucleic acids by subjecting the double-stranded target mutant and
wild-type nucleic acids to a first denaturing temperature that is
above the melting temperature of the wild-type nucleic acid;
(b) forming a target mutant/wild-type strand duplex;
(c) denaturing said mutant/wild-type strand duplex by subjecting
the nucleic acid sample to a critical temperature (Tc) that is
below the Tm of the wild-type nucleic acids;
(d) annealing a primer pair to the mutant and wild-type target
nucleic acid strands; and
(e) extending said primer pair so as to enrich said mutant target
sequence relative to said wild-type strand.
In some embodiments, COLD-PCR is performed while subjecting the
provided contents in the single reaction vessel to an amplification
condition under which the set of tail primers anneal (e.g., during
step (d)).
In some embodiments, COLD-PCR is performed after subjecting the
provided contents in the single reaction vessel to an amplification
condition under which the set of tail primers anneal (e.g., after
step (d)). In some embodiments, COLD-PCR is performed for 1-50
cycles (e.g., 1-40, 2-30, 5-25, 8-20 or 5-10 cycles) to enrich
mutant target nucleic acid relative to wild-type target nucleic
acid.
In some embodiments, COLD-PCR is performed in the same tube as the
amplification methods described herein. In some embodiments, the
reagents for the enrichment of mutant alleles of the target nucleic
acids relative to wild-type alleles of the target nucleic acids are
provided with the double-stranded DNA (e.g., genomic DNA, or cDNA),
the set of outer multiplexed primers, the set of inner multiplexed
primers and the set of tail primers in the single reaction
vessel.
If the above example of COLD-PCR were to be adapted for oscillating
COLD-PCR, steps (b) and (c) would be repeated. In some embodiments
of oscillating COLD-PCR, forming a target mutant/wild-type strand
duplex and denaturing said mutant/wild-type strand duplex, is
repeated 1-29 times (e.g., 1-19 or 2-9 times).
Methods of ice-COLD-PCR and TT-COLD-PCR are described in WO
2012/135664, which is incorporated by reference herein in its
entirety. A non-limiting example of a ice-COLD-PCR protocol
includes:
(a) exposing the mutant and wild-type target nucleic acids to a
reference sequence that is complementary the target sequence;
(b) denaturing the double-stranded target mutant and wild-type
nucleic acids by subjecting the double-stranded mutant and
wild-type target nucleic acids to a first denaturing temperature
that is above the melting temperature of the wild-type nucleic
acid;
(c) forming a target mutant/reference strand and target
wild-type/reference strand duplexes;
(d) denaturing said mutant/reference strand duplex by subjecting
the nucleic acid sample to a critical temperature (Tc) that is
below the Tm of the wild-type/reference duplex;
(e) annealing a primer pair to the mutant and wild-type target
nucleic acid strands; and
(f) extending said primer pair so as to enrich said mutant target
sequence relative to said wild-type target nucleic acid.
A non-limiting example of a TT-ice-COLD-PCR (also known as
temperature independent (TI)-ice-COLD-PCR) protocol includes:
(a) exposing the mutant and wild-type target nucleic acids to a
reference sequence that is complementary the target sequence;
(b) denaturing the double-stranded target mutant and wild-type
target nucleic acids by subjecting the double-stranded target
mutant and wild-type nucleic acids to a denaturing temperature that
is above the melting temperature of the wild-type nucleic acid;
(c) forming a target mutant/reference strand and target
wild-type/reference strand duplexes;
(d) denaturing said mutant/reference strand duplex by subjecting
the nucleic acid sample to a first critical temperature (Tc) that
is below the Tm of the wild-type/reference duplex;
(e) annealing a primer pair to the mutant and wild-type target
nucleic acid strands;
(f) extending said primer pair so as to enrich said mutant target
nucleic acid relative to said wild-type target nucleic acid;
and
(f) repeating steps (d) to (f) at least once at a second Tc which
is above the first Tc.
In some embodiments, any form of COLD-PCR (as described above) with
or without amplification results in mutation enrichment relative to
wild-type target nucleic acids of 1-200-fold (e.g., 1-150-, 5-100-
or 10-100-fold) compared to the unenriched sample. In some
embodiments, any form of COLD-PCR with or without amplification
results in mutation enrichment relative to wild-type target nucleic
acids of more than 200-fold (e.g., 250-fold or 300-fold) compared
to the unenriched sample.
In some embodiments, DiSSECT is used to enrich mutant target
nucleic acids relative to wild-type target nucleic acids. DiSSECT
is a method that enriches unknown mutations of targeted DNA
sequences purely based on thermal denaturation of DNA duplexes
without the need for enzymatic reactions. Methods of DiSSECT are
described Guha et al. (Nucleic Acids Research, 2012, 1-9), which is
incorporated herein by reference in its entirety. A non-limiting
example of a DiSSECT protocol includes:
(a) allowing mutant and wildtype target nucleic acids to bind to
complementary probes which are immobilized to beads, wherein the
probes resemble the wild-type nucleic acids;
(b) denaturing the target mutant/probe duplex by subjecting the
nucleic acid sample to a critical temperature such that the
wild-type/probe duplex does not denature;
(c) collecting the eluate from the beads; and
(d) repeating at least once (a)-(c) using beads on which the probes
are unbound to any nucleic acid.
In some embodiments, DiSSECT is performed for 1-20 cycles (e.g.,
1-18, 2-6, 2-4, 2-10 or 5-15 cycles). In some embodiments, DiSSECT
results in mutation enrichment relative to wild-type target nucleic
acids of 1-600-fold (e.g., 100-fold, 200-fold, 300-fold, 400-fold,
500-fold or 600-fold) compared to the unenriched sample.
Toehold PCR is described by Wu et al. Nat Methods. 2015 December;
12(12):1191-6, which is incorporated herein in its entirety.
The term `mutant` refers to a nucleotide change (i.e., a single or
multiple nucleotide substitution, deletion, insertion, or
methylation, or alteration in the number of poly-nucleotide
repeats) in a nucleic acid sequence. A nucleic acid which bears a
mutation has a nucleic acid sequence (mutant allele) that is
different in sequence from that of the corresponding wild-type
sequence. Herein, the term "mutant target nucleic acid" is used
interchangeably with "mutant alleles of target nucleic acid."
Similarly, the term "wild-type target nucleic acid" is used
interchangeably with "wild-type alleles of target nucleic acid."
The mutant alleles can contain between 1 and 500 nucleotide
sequence changes. A mutant allele may have 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,
50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nucleotide sequence
changes compared to a corresponding wild-type allele. Typically, a
mutant allele will contain between 1 and 10 nucleotide sequence
changes, and more typically between 1 and 5 nucleotide sequence
changes. The mutant allele will have 50%, 60%, 70%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the
wild-type allele. Generally, the mutant allele will be obtained
from diseased tissues or cells and is associated with a disease
state.
`Allele` refers to alternative forms of a gene, portion thereof or
non-coding region of DNA that occupy the same locus or position on
homologous chromosomes that have at least one difference in the
nucleotide sequence. The term allele can be used to describe DNA
from any organism including but not limited to bacteria, viruses,
fungi, protozoa, molds, yeasts, plants, humans, non-humans,
animals, and archaebacteria. The alleles may be found in a single
cell (e.g., two alleles, one inherited from the father and one from
the mother) or within a population of cells (e.g., a wild-type
allele from normal tissue and a somatic mutant allele from diseased
tissue). Alleles will generally share 50%, 60%, 70%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to
each other.
Quantitation
Low-level tumor somatic DNA mutations can have profound
implications for development of metastasis, prognosis, choice of
treatment, follow-up or early cancer detection. Unless effectively
detected, these low-level mutations can misinform patient
management decisions or become missed opportunities for
personalized medicine. Next generation sequencing (NGS)
technologies reveal prevalent somatic mutations, yet they `lose
steam` when it comes to detecting low-level DNA mutations in tumors
with clonal heterogeneity, or in bodily fluids during `liquid
biopsy`, and their integration with clinical practice is not
straightforward. For mutations at an abundance of .about.2-5% or
less, NGS generates false positives (`noise`) independent of
sequencing depth and hinders personalized clinical decisions based
on mutational profiling. Recent enhancements employing single
molecule barcoding (or Unique Identifiers, UIDs) enable NGS to
overcome noise and detect `ultra-rare mutations`. (Kinde et al.,
Proc Natl Acad Sci USA 2011, 108:9530-5; Schmitt et al., Proc Natl
Acad Sci USA 2012; Gregory et al., Nucleic Acids Res 2016, 44:e22;
Jee et al., Nature 2016). Furthermore, the use of molecular
barcodes (UIDs) at the initial stages of sample preparation (i.e.
before application of mutation enrichment via COLD-PCR or NaME-PrO)
allows for strict quantification of original mutation abundance
following mutation enrichment.
One exemplary method of quantifying mutant DNA using barcodes in
conjunction with the amplification methods described herein is
shown in FIG. 12. In step 1, the DNA (e.g., genomic DNA, or cDNA)
is fragmented. In step 2, barcodes, e.g., unique identifiers, with
upstream common sequence tags, are ligated onto both ends of the
fragmented DNA. The box outlines the amplification methods
described herein. In the first amplification reaction, the
fragmented DNA is amplified with an outer forward primer
complimentary to the common sequence tag (e.g., a tail) and a
gene-specific outer reverse primer. This generates a DNA fragment
with a barcode and tail on one end. In the second amplification
reaction, the product of the first amplification reaction is
amplified with an inner forward primer complimentary to the common
sequence tag and an inner reverse primer that has a gene-specific
portion that is nested relative to the outer reverse primer, and
that further comprises a tail. This generates a DNA molecule with a
tail and barcode on one end and a tail on the other end. In the
third amplification reaction, the product of the second
amplification reaction is amplified with a forward tail primer that
is complimentary to the common sequence tag and a reverse tail
primer that is complementary to the tail of the inner reverse
primer. This generates a DNA molecule with a tail and barcode on
one end and a tail on the other end. The mutant allele can either
be enriched by performing COLD-PCR in conjunction with the
amplification reaction, as is described above, or by performing
NaME-PrO enrichment after the amplification reaction, followed by
10 cycles of PCR using the tail primers.
The term "barcode" as used herein refers to a unique sequence of
nucleotides that allows identification of the nucleic acid of which
the barcode is a part. Barcoding a DNA fragment is a process by
which the DNA fragment is uniquely tagged with one or more short
identifying sequences. In some embodiments, it is desired for each
DNA fragment in a sample to have a barcode that is unique from
barcodes on any other DNA fragment in the sample. In some
embodiments, each DNA fragment in a sample comprises one unique
barcode. In some embodiments, each DNA fragment in as sample
comprises two barcodes that are unique from each other and unique
from any other barcode that is attached to any other DNA fragment
in the sample. Such uniqueness of barcodes in a sample of DNA
fragments can be accomplished, for example, by optimizing the
length of each barcode (i.e., the number of nucleotides in each
barcode) and/or the ratio of unique barcodes to DNA fragments
during barcoding (i.e., attaching barcodes to DNA fragments).
Barcodes can be any appropriate length. In some embodiments, the
length of each barcode used to barcode DNA fragments in a sample is
6-20 bp long (e.g., 8-18 bp, 8-14 bp, 10-16 bp or 12-14 bp). In
some embodiments, the length of each barcode used to barcode DNA
fragments in a sample is 14 bp long.
Barcodes can be attached to DNA fragments in any appropriate ratio.
In some embodiments, the ratio of unique barcodes to DNA fragments
during barcoding is 10.sup.6-10.sup.10 (e.g., 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, 10.sup.10) unique barcodes to 100 ng of DNA (or
3.times.10.sup.4 allelic copies).
Methods of attaching barcodes to nucleic acids are known in the
art. Various publications provide descriptions of barcoding
technology. For example, Wong and Moqtaderi (Curr Protoc Mol Biol.
2013; Chapter 7:Unit 7.11) describe a barcoding protocol for the
preparation of up to 96 ChIP samples for multiplex sequencing in a
single flow cell lane on the Illumina platform; and Stahlberg et
al. (Nucleic Acids Res. 2016 Jun. 20; 44) describe a PCR-based
barcoding method, both of which are incorporated herein by
reference in their entirety. The following patents that also
describe DNA barcoding methods are also incorporated herein by
reference in their entirety: U.S. Pat. Nos. 8,691,509, 8,268,564
and US application 20120220494A1.
In some embodiments, double-stranded barcodes are attached to a
double-stranded DNA fragment by ligation. In some embodiments,
double-stranded DNA fragments are barcoded using PCR technique
employing primers that comprise unique barcodes.
In some embodiments, the barcodes are attached to the DNA (e.g.,
genomic DNA, or cDNA) with a common sequence tag prior to the
amplification methods described herein. As used herein, the term,
"common sequence tag" refers to a nucleotide sequence that is
common to all the DNA fragments in a sample, e.g., a sample of
genomic fragmented DNA. A common tag enables processing of all the
DNA fragments in a sample. For example, primers complementary to a
common tag in a sample may be used to amplify all DNA fragments in
a sample, regardless of whether a DNA fragment contains target
nucleic acid (e.g., mutant target nucleic acid or wild-type target
nucleic acid) or non-target nucleic acid.
In some embodiments, the common sequence tag comprises the forward
or reverse tail sequence. In some embodiments, the outer forward
primer comprises a common sequence tag. In some embodiments, the
outer reverse primer comprises a common sequence tag. In some
embodiments, the inner forward primer comprises a common sequence
tag. In some embodiments, the inner reverse primer comprises a
common sequence tag. In some embodiments, the forward tail primer
comprises a common sequence tag. In some embodiments, the reverse
tail primer comprises a common sequence tag.
For any one of the methods disclosed herein, a sample of
double-stranded DNA (e.g., genomic DNA, or cDNA) may comprise
double-stranded DNA fragments, wherein each terminus of the DNA
fragments is attached to a unique double-stranded barcode and a
double-stranded common sequence tag, wherein the common sequence
tag is located upstream of the unique barcode. By being located
"upstream" of the unique tag, it is meant that the common tag is
located 5' relative to the unique tag if the unique barcode, common
tag and DNA fragment sequence are read from 5' to 3'.
Common sequence tags can be any appropriate length. In some
embodiments, a common tag is 16-40 bp long (e.g., 16-40, 18-36,
20-32, 22-30 or 24-28 bp long). In some embodiments, a common tag
is 18 nucleotides long (i.e. an 18-mer). It is to be understood
that a the terms "nucleotide" and `base pair (bp)" are used
interchangeably herein.
In some embodiments, a unique barcode and a common tag are attached
to each end of a double-stranded DNA fragment at the same time
using the same method. In some embodiments, a barcode and a common
tag are attached to each end of a double-stranded DNA fragment by
ligation.
In some embodiments, a unique barcode and common tag are attached
to a terminus of a DNA fragment by starting from a single-stranded
barcode, synthesizing the opposite strand of the single-stranded
barcode using an extension reaction to form a double stranded
barcode, and the ligating and end of the double-stranded DNA (e.g.,
genomic DNA, or cDNA) fragment to the end of the barcode.
In some embodiments, a barcode and a common tag are attached to
each end of a double-stranded DNA fragment by using
multiplexed-PCR. In such embodiments, PCR using oligonucleotide
primers are used, wherein each oligonucleotide primer comprises a
common tag portion, a unique barcode portion and a target-specific
portion. The target-specific portion enables attachment of the
oligonucleotide primer to anneal to DNA fragments.
In some embodiments, the methods disclosed herein require DNA
(e.g., genomic DNA, or cDNA) to be in fragmented form In some
embodiments, DNA in a sample collected from a subject is already
fragmented. For example, a sample of cell-free DNA or DNA
circulating in blood is fragmented when collected. In some
embodiments, DNA from samples of the urine of a subject is
fragmented. In some embodiments, DNA collected from bodily fluid or
tissue sample of a subject is not fragmented and needs to be
fragmented. In some embodiments, a sample of DNA is fragmented but
it is desired to fragment it further to make smaller fragments.
Various techniques to fragment double-stranded DNA are known in the
art. In some embodiments, DNA is sheared physically (e.g., using
acoustic shearing using a Covaris instrument, sonication using a
Bioruptor or hydrodynamic shearing using a Hydroshear instrument).
In some methods, double-stranded DNA is sheared enzymatically using
any DNAase type of enzyme that digests DNA randomly (e.g., a
Shearase, DNAse1 or a transposase). In some embodiments,
double-stranded DNA is fragmented by chemical fragmentation (e.g.,
by exposing the DNA to be fragmented to heat and divalent metal
cation. Depending on the method of DNA fragmentation, DNA fragments
may be subjected to enzymatic end-repairing to obtain blunt
ends.
In some embodiments of any one of the methods disclosed herein, a
double-stranded DNA fragment is 20-400 bp long (e.g., 10-400,
40-200, 50-150 or 50-100 bp long).
In any of the methods disclosed herein, obtaining a measure of
total unique barcodes in a sample may be accomplished using DNA
sequencing methods. Several sequencing methods and protocols for
sample preparation for these methods are well-established in the
art. Indeed, one of the advantages of the methods disclosed herein
is that they are compatible with established methods of sample
preparation for sequencing methods used in the field. Examples of
methods of sequencing include SANGER sequencing, MiSeq sequencing,
massively parallel signature sequencing (MPSS), polony sequencing,
454 sequencing, Illumina (or Solexa) sequencing, SoLiD sequencing,
Ion Torrent semiconductor sequencing, single molecule real time
(SMRT) sequencing, and nanopore sequencing. The following
publications describe various sequencing options and are
incorporated herein by reference in their entirely: Goodwin et al.
(Coming of age: ten years of next-generation sequencing
technologies, Nature Reviews Genetics 17, 333-351 (2016)), Heather
and Chain (The sequence of sequencers: The history of sequencing
DNA, Genomics, 107: 1-8 (2016)), and Moorthie et al. (Review of
massively parallel DNA sequencing technologies, Hugo J. 2011
December; 5(1-4): 1-12).
EXAMPLES
Example 1: Combined Multiplexed-PCR Reactions that Provide Target
Enrichment and Amplification Along with Mutation Enrichment
The processes of target enrichment and mutation enrichment can be
combined in a single tube reaction (`all-in-one`) to combine the
sequential steps of selecting DNA targets for sequencing from DNA
(e.g., genomic DNA, or cDNA), and addition of the Illumina
sequencing adaptor. This highly efficient, all-in-one reaction
includes in a single tube: 1. The DNA to be interrogated (genomic
DNA, circulating DNA, saliva DNA or DNA from any other source
human, animal of plant). 2. A first set of multiplexed, outer
primers that target the DNA sites of interest 3. A second set of
inner multiplexed primers that are nested to the outer primers.
These primers comprise a gene-specific portion and common forward
and reverse oligonucleotide `tails` towards the 5'end. The tails
optionally comprise 20-30 bp of the 3'-end portion of the Illumina
adaptors that enable binding to the Illumina flow-cell during
sequencing. Or similarly, to enable binding to the Qiagen
sequencing adaptors; or binding to the Ion Torrent sequencing cell;
or adaptors to the sequencing cell of any other sequencing system.
4. A forward and a reverse oligonucleotide `tail`, without the
gene-specific portion, which can be used as a primer to amplify all
sequences carrying the same tail on their 5'end. 5. PCR
amplification components: DNA polymerase, dNTP, PCR buffer to
enable PCR amplification
The all-in-one reaction approach is described in FIG. 1. The
multi-stage single tube PCR reaction can optionally start with a
low number of cycles (e.g., 10 cycles) to produce an initial
amplification of selected targets from genomic DNA using an
annealing temperature (Ta) that fits the outer multiplexed primers.
Similarly, the `tail` primers will not participate in the reaction
as there is no corresponding binding site (for example, the tails
may have the sequence corresponding to the 3'-end of the Ilumina
sequencing adaptor, which has no homology to common human
sequences).
Following the first 10 PCR cycles, there will be enough target
built-up from the outer multiplexed primers that the anchor-tail
primers can bind to it and generate nested PCR product carrying the
sequence of the `tails` on the 5'end. The annealing temperature can
also be adjusted to fit optimally the annealing temperature of the
anchor-tail oligonucleotides. Because of their very low
concentration (0.001-0.01 .mu.M), and different Ta, these
oligonucleotides do not produce significant amount of product
directly from the original genomic DNA during the initial 10
cycles. Similarly, the `tail` primers do not participate in the
reaction during the first 10 cycles as there is no corresponding
binding site in genomic DNA (for example, the tails may have the
sequence corresponding to the 3'-end of the Ilumina sequencing
adaptor, which has no homology to common human sequences).
However, once the pre-amplification reaction builds enough product
following the initial 10 cycles, then anchor-tail oligonucleotides,
which are nested to the outer oligonucleotides, start generating
products containing the Tails (tail 1=forward; tail 2=reverse). The
tails are at high concentration (0.1-0.2 .mu.M) such that, in
subsequent cycles, they take over the amplification for the
remaining 20-30 cycles of the reaction.
In this way, the `all-in-one reaction` enables highly specific
selection of DNA targets (in view of two nested PCR reactions), and
incorporation of a tail sequence that corresponds to the Illumina
sequencing adaptor. Finally, to prepare the product for sequencing,
an additional few cycles of PCR using the Illumina adaptor
containing individual `sample barcodes` can be used and the product
is processed for sequencing.
In FIG. 1 described above, it is also note-worthy that (optionally)
to separate the action of the first set of (outer) multiplexed
primers from the action of the second set (nested) anchor-tail
primers within the multi-stage PCR reaction, a substantially
different annealing temperature Ta can be used for these two sets
of primers. For example, the Tm of the multiplexed outer primers
can be 65.degree. C., so that a Ta of 60-65.degree. C. would be
appropriate for these primers; while the Tm of the anchor-tail
primers can be 55.degree. C. so that a Ta of 50-55.degree. C. would
be appropriate. In this example, during the first 10 cycles of PCR
using Ta=65.degree. C. mainly the outer primers would generate
product from genomic DNA. And by changing the Ta to 50.degree. C.
after the first 10 cycles one would activate the nested multiplexed
anchor-MB-tail primers to generate highly specific, nested products
using the amplicons generated by the outer primers.
Hot Start Primers
As an additional way to separate the action of the various primers
within the multi-stage PCR reaction, one may include `hot start
primers` (available from Trilink Technologies, Inc.), whose action
is only activated when the temperature stays at high levels (e.g.,
90-95.degree. C. for 2-10 min). In this way the thermo-activatable
primers will not interfere at earlier steps of the `all-in-one`
reaction, and will only become activated at a selected time point.
For example, the anchor-tail primers can be designed as `hot`
start` primers using a 3-end modification provided by Trilink Inc.
Thus, after the first 10 cycles of PCR, the temperature can be
elevated for a few minutes to 95.degree. C. to activate the second
set of primers. In the same way, a `hot start` tail can be used to
regulate when the tail primer can be activated in the reaction.
Thus, one can arrange that the tail is activated after the initial
14 cycles of PCR, upon which both the outer and the inner primers
have applied their actions and produced the required template for
the tail primers. Finally, one may also include host start versions
of the Illumina adaptors shown at the bottom of FIG. 1, in the
multi-stage PCR reaction from the start. The Illumina adaptors will
only become thermo-activated after all the other primers included
in the reaction complete their intended action. In this way, there
will not be a need for an additional PCR to attach the Illumina
adaptors at the end.
An example of the all-in-one reaction for multiplexed PCR with 54
target genes co-amplified from circulating DNA is shown on FIG. 2.
The protocol described in FIG. 1 was applied for the 54 gene
targets shown on the x-axis, while the y axis shown the number of
sequencing reads obtained for each of the targets tested. The
all-in-one protocol in FIG. 1 (in BLACK) is compared to a
conventional step-by-step multiplexed PCR approach, during which
the outer primers are used for a first standard multiplexed PCR;
this is then followed with sample purification and dilution and a
second, nested PCR (in WHITE). Both approaches show the same
general trends, thus validating the single step approach.
All-in-One, Single Tube PCR Reaction Incorporating Mutation
Enrichment Via COLD-PCR
The methods described above may optionally also incorporate
COLD-PCR cycling during the last part of the multi-stage PCR
reaction, thereby providing selective amplification of
mutation-containing sequences in addition to highly specific target
selection. This approach is shown in FIG. 3, in combination with
ICE-COLD-PCR. In this approach, a blocker Reference Sequence is
included in the reaction mix from the beginning, and the approach
shown in FIG. 1 is now modified to incorporate ICE-COLD-PCR cycling
for the last 10-15 cycles of the reaction.
An example of an all-in-one, single step reaction incorporating
mutation enrichment for two targets, KRAS and BRAF, is shown in
FIG. 4 (PCR cycling conditions), FIG. 5 (results of testing KRAS
mutation enrichment in serial mutation dilutions via ddPCR) and
FIG. 6 (results of testing BRAF mutation enrichment in serial
mutation dilutions via ddPCR). The data show that strong mutation
enrichment occurs simultaneously with highly specific target
selection in a single reaction from genomic DNA.
All-in-One, Single Tube PCR Reaction Incorporating Mutation
Enrichment Via NaME-PrO
The methods described above may optionally also incorporate
NaME-PrO reaction during the first step of the process, followed by
a multi-stage all-in-one PCR reaction, thereby providing selective
amplification of mutation-containing sequences in addition to
highly specific target selection, FIG. 7. NaME-PrO is applied
directly on genomic DNA as described.sup.5.
All-in-One, Single Tube PCR Reaction Incorporating Molecular
Barcodes Plus Optional Mutation Enrichment Via NaME-PrO (OR Via
COLD-PCR)
The methods can also be combined to enable an all-in-one reaction
that retains the molecular barcode UID on one end of the targets,
as described in FIG. 8. Enrichment can be provided either via
COLD-PCR protocol at the last several cycles of the all-in-one
reaction described in step 3 OR by an additional NAME-PRO step plus
amplification/library construction. The general methods described
in previous sections can be applied here too, in order to achieve
all-in-one target selection, mutation enrichment and molecular
barcode retention so that the UID can be sequenced along with the
selected targets.
LITERATURE CITED
[1] Kinde I, Wu J, Papadopoulos N, Kinzler K W, Vogelstein B:
Detection and quantification of rare mutations with massively
parallel sequencing. Proc Natl Acad Sci USA 2011, 108:9530-5. [2]
Schmitt M W, Kennedy S R, Salk J J, Fox E J, Hiatt J B, Loeb L A:
Detection of ultra-rare mutations by next-generation sequencing.
Proc Natl Acad Sci USA 2012. [3] Gregory M T, Bertout J A, Ericson
N G, Taylor S D, Mukherjee R, Robins H S, Drescher C W, Bielas J H:
Targeted single molecule mutation detection with massively parallel
sequencing. Nucleic Acids Res 2016, 44:e22. [4] Jee J, Rasouly A,
Shamovsky I, Akivis Y, S R S, Mishra B, Nudler E: Rates and
mechanisms of bacterial mutagenesis from maximum-depth sequencing.
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Kulke M H, Makrigiorgos G M: Elimination of unaltered DNA in mixed
clinical samples via nuclease-assisted minor-allele enrichment.
Nucleic Acids Res 2016. [6] Shagin D A, Rebrikov D V, Kozhemyako V
B, Altshuler I M, Shcheglov A S, Zhulidov P A, Bogdanova E A,
Staroverov D B, Rasskazov V A, Lukyanov S: A novel method for SNP
detection using a new duplex-specific nuclease from crab
hepatopancreas. Genome research 2002, 12:1935-42. [7] Gnirke A,
Melnikov A, Maguire J, Rogov P, LeProust E M, Brockman W, Fennell
T, Giannoukos G, Fisher S, Russ C, Gabriel S, Jaffe D B, Lander E
S, Nusbaum C: Solution hybrid selection with ultra-long
oligonucleotides for massively parallel targeted sequencing. Nat
Biotechnol 2009, 27:182-9. [8] Mertes F, Elsharawy A, Sauer S, van
Helvoort J M, van der Zaag P J, Franke A, Nilsson M, Lehrach H,
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10:374-86.
OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined
in any combination. Each feature disclosed in this specification
may be replaced by an alternative feature serving the same,
equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
From the above description, one skilled in the art can easily
ascertain the essential characteristics of the present invention,
and without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions. Thus, other embodiments are also
within the claims.
EQUIVALENTS
While several inventive embodiments have been described and
illustrated herein, those of ordinary skill in the art will readily
envision a variety of other means and/or structures for performing
the function and/or obtaining the results and/or one or more of the
advantages described herein, and each of such variations and/or
modifications is deemed to be within the scope of the inventive
embodiments described herein. More generally, those skilled in the
art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the inventive teachings is/are used. Those
skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
inventive embodiments described herein. It is, therefore, to be
understood that the foregoing embodiments are presented by way of
example only and that, within the scope of the appended claims and
equivalents thereto, inventive embodiments may be practiced
otherwise than as specifically described and claimed. Inventive
embodiments of the present disclosure are directed to each
individual feature, system, article, material, kit, and/or method
described herein. In addition, any combination of two or more such
features, systems, articles, materials, kits, and/or methods, if
such features, systems, articles, materials, kits, and/or methods
are not mutually inconsistent, is included within the inventive
scope of the present disclosure.
All definitions, as defined and used herein, should be understood
to control over dictionary definitions, definitions in documents
incorporated by reference, and/or ordinary meanings of the defined
terms.
All references, patents and patent applications disclosed herein
are incorporated by reference with respect to the subject matter
for which each is cited, which in some cases may encompass the
entirety of the document.
The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
The phrase "and/or," as used herein in the specification and in the
claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, "or" should
be understood to have the same meaning as "and/or" as defined
above. For example, when separating items in a list, "or" or
"and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
As used herein in the specification and in the claims, the phrase
"at least one," in reference to a list of one or more elements,
should be understood to mean at least one element selected from any
one or more of the elements in the list of elements, but not
necessarily including at least one of each and every element
specifically listed within the list of elements and not excluding
any combinations of elements in the list of elements. This
definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the
contrary, in any methods claimed herein that include more than one
step or act, the order of the steps or acts of the method is not
necessarily limited to the order in which the steps or acts of the
method are recited.
* * * * *
References